Registration Dossier
Registration Dossier
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EC number: 601-779-5 | CAS number: 121451-02-3
Endpoint summary
Administrative data
Description of key information
Subchronic toxicity (oral): LOEL and NOAEL were 1.0 mg/kg/day based on an increase in relative liver weights (male and female rats) and microscopic hepatocellular hypertrophy in males. EPA OPPTS 87.4300, EPA OPPTS 870.3100, OECD 453, OECD 408, EU Method Part B, EU Method B.26, and JMAFF guidelines, Yano & Dryzga (2002)
Chronic toxicity (oral): EPA OPPTS 87.4300 and 870.3100, OECD 453 and 408, EU Method B.26, and JMAFF. Yano et al (2005)
-90 day NOEL 1.0 mg/kg bw/day based on increased relative kidney weight and absolute and relative ovary weights of females given 1.0 mg/kg bw/day.
- 1 year NOEL 1.0 mg/kg bw/day based on decreased body weight/gains and increased liver weights in males and females, increased kidney weights (males only), and increased adrenal weights (females only) observed at 75 and 300 mg/kg bw/day. Further effects included of hepatocellular hypertrophy (males only), lung inflammation and in males given 300 mg/kg/day two hepatocellular adenomas.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 24 July 1997 - 18 August 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The palatability of the test material was determined in female Beagle dogs (2/dose group), which were offered diets formulated with the test material at 1.5 or 3.0 % in certified canine diet for approximately 14 days. Observation included; clinical signs, feed consumption, body weight and body weight gain. Animals were necropsied at the end of the study.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Housing: Individually in runs.
- Diet: Certified canine diet, provided ad libitum.
- Water: Pressurized water source, provided ad libitum. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Two weeks (14 consecutive days).
- Frequency of treatment:
- Daily.
- Dose / conc.:
- 15 000 mg/kg diet
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 30 000 mg/kg diet
- Remarks:
- Basis:
nominal in diet - No. of animals per sex per dose:
- Two females per dose group.
- Control animals:
- no
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Recorded twice daily.
CLINICAL EXAMINATION: Yes
- Time schedule: Performed weekly.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured pre-test and then on days 2, 4, 8, 11 and 15.
FOOD CONSUMPTION
- Food consumption for each animal determined as g food/day: Yes
Food consumption was calculated pre-test and then on days 2, 4, 8, 11 and 15. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, all animals were necropsied at termination.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Mortality:
- no mortality observed
- Description (incidence):
- No treatment related effects.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Soft faeces were observed sporadically in the dogs fed diet containing the test material at 3.0 % both pre-test and during weeks 1 and 2 and therefore were not considered to be treatment related. There were no abnormal clinical signs observed at the 1.5 % dose level.
BODY WEIGHT AND WEIGHT GAIN
All animals gained between 6 and 10 % body weight over the two week study period.
FOOD CONSUMPTION
Measured feed consumption for the animals given diet containing the test material at 1.5 % diet was lower during the dosing period than for the pre-test interval, however, this difference was interpreted as being due to the abnormally high pre-test feed consumption values (historical feed consumption is 306 g/day for female control dogs).
GROSS PATHOLOGY
A pituitary cyst found at the 1.5 % dose level was the only gross necropsy finding and does not appear to be treatment related. - Basis for effect level:
- other: A NOEL was not determined in this preliminary palatability study.
- Remarks on result:
- not measured/tested
- Remarks:
- Effect level not specified
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, diet containing the test material at either 1.5 % or 3.0 % would be sufficiently palatable to dogs based on measured feed consumption and associated body weight gain. The test showed that these concentrations could be used in a subsequent dietary toxicity study.
- Executive summary:
The palatability of the test material in certified canine diet was assessed in a preliminary study conducted in accordance with sound scientific principles. Female Beagle dogs (2/dose group) were offered diets formulated with the test material at 1.5 or 3.0 % in certified canine Lab Diet for approximately 14 days in a non-GLP study.
No treatment related clinical observations were observed at either dose level. The diet appeared to be palatable at both dose levels and all animals gained between 6 and 10 % body weight over the two week study period. Measured feed consumption for the animals given diet containing the test material at 1.5 % was lower during the dosing period than for the pre-test interval, however, this difference was interpreted as being due to the abnormally high pre-test feed consumption values (historical feed consumption is 306 g/day for female control dogs). Thus, these data suggest that a diet containing the test material at either 1.5 % or 3.0 % would be sufficiently palatable to the dog and that these concentrations could be used in a subsequent dietary toxicity study.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 17 July 1997 - 08 October 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6-8 weeks at study start.
- Housing: Individually in stainless steel cages.
- Diet: Rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: The animals were housed two per cage in stainless steel wire cages and acclimated to the laboratory for at least one week prior to study start day.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): average 22.1 °C (ranged from 22 - 24 °C).
- Humidity (%): mean 51.7 % (ranged from 47 - 61 %).
- Photoperiod (hrs dark / hrs light): An adequate photocycle was maintained.
IN-LIFE DATES: From: 17 July 1997 To: 14 August 1997 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Premixes and diets were prepared weekly during the study.
- Mixing appropriate amounts with (Type of food): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed according to the SOPs of the Oral/Dermal Toxicity Section of the Toxicology Research Laboratory. Each premix was prepared by alternating layers of ground feed and premix/test material concentrate and mixing for a pre-determined amount of time using a paddle-type mixer. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 4 consecutive weeks.
- Frequency of treatment:
- Daily, 7 days per week.
- Remarks:
- Doses / Concentrations:
0, 1, 10, 100, 500 or 1000 mg/kg/day
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 1.0, 10.4, 101.4, 512.6, and 1029.1 mg/kg/day
Basis:
other: Calculated actual male dosages - Remarks:
- Doses / Concentrations:
0, 1.1, 10.9, 105.1, 520.6, and 1055.6 mg/kg/day
Basis:
other: Calculated actual female dosages - No. of animals per sex per dose:
- Five per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high dose level of 1000 mg/kg bw/day represented a limit test dosage, as specified by several regulatory agencies for dietary studies. The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL) based on the previous toxicity data.
- Previous toxicity data Oral LD50 > 2000 mg/kg bw (female Fischer 344 rats) and dermal LD50 > 2000 mg/kg bw (rabbit).
- Dose route rationale: Probable routes of human exposure to the test material would be by ingestion of foodstuffs that may contain low levels of residues or from accidental ingestion during field application or manufacture. Thus, administration of the test material via the diet for 7 days per week represented an appropriate means of evaluating systemic exposure to animals.
- Rationale for animal assignment: Rats were assigned to groups using a computer-generated randomisation program based on body weights - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, made each day of the work week. A cageside examination was not conducted on the days a clinical examination was performed, since the clinical examination was more thorough.
- Observations: This examination included careful, hand-held evaluations of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behaviour.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were conducted on all animals prior to the start of the study and weekly throughout the study period.
- Observations: This examination included careful, hand-held evaluations of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behaviour.
ADDITIONAL IN LIFE OBSERVATIONS: Yes
- Time schedule: An additional observation was made each day of the work week, as well as twice daily on weekends.
- Observations: Moribundity, mortality, and the availability of feed and water.
BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-dosing period and at weekly intervals during the dosing periods. Terminal body weights on fasted rats were recorded.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/ day: Yes
Feed consumption data were collected weekly for all animals. Feeders were weighed at the start and end of a measurement cycle, and consumption was calculated using the following equation.
Feed consumption (g/day) = (initial weight of feed crock – final weight of feed crock) / (No. of days in measurement cycle)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Time weighted average feed intake (mg/kg/day) = [(feed consumption * 1000) * (% test material in diet/1000)] / {[[(BWf – BWj) / (Dayf – Dayi)] * [(Dayf – Dayi)/2] * + BWi] / 1000}
Where:
BWf = final body weight of measurement cycle
BWi = initial body weight of measurement cycle
Dayi = day of study on which initial body weight was collected
Dayf = day of study on which final body weight was collected
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Collected at time of necropsy.
- Anaesthetic used for blood collection: Yes (methoxyflurane).
- Animals fasted: Yes, fasted overnight.
- How many animals: All animals.
- Method and sample preparation: Blood samples were collected via puncture of the orbital sinus. Samples for haematology were mixed with ethylenediamine-tetraacetic acid anticoagulant and blood smears were prepared and stained with Wright’s stain.
- Parameters checked: The following parameters were assayed using a Technicon H-1E Haematology Analyzer: haematocrit (HCT), haemoglobin concentration (HGB), erythrocyte count (RBC), leukocyte count (WBC), platelet count (PLAT) differential counts of 100 leukocytes, morphology of erythrocytes, leukocytes and platelets and RBC indices. Methaemaglobin was assayed via a colometric method using a Beckman DU62 Spectrophotometer according to the SOP’s of the Pathology Section of the Toxicology Research Laboratory.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Collected at time of necropsy.
- Anaesthetic used for blood collection: Yes (methoxyflurane).
- Animals fasted: Yes, fasted overnight.
- How many animals: All animals.
- Method and sample preparation: Blood samples were collected via puncture of the orbital sinus. Sample for clinical chemistry were held on ice and serum was harvested.
- Parameters checked: The following parameters were assayed using a Hitachi 914 Clinical Chemistry Analyzer: concentrations of urea nitrogen (UN), creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLOB), cholesterol (CHOL), triglycerides (TRIG), total bilirubin (TBIL), glucose (GLUC), and electrolytes (Ca, Na, K, Cl); enzyme activities of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and creatine phosphokinase (CK).
URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from all animals by manual compression of the abdomen two days prior to the scheduled necropsy.
- How many animals: All animals.
- Parameters checked: The urinalysis, consisted of determination of urine specific gravity (refractometer), a qualitative assessment of colour and appearance, a microscopic examination of the urine sediment, and a semi-quantitative determination of: pH, bilirubin, glucose, proteins, ketones, occult blood and urobilinogen (Mulistix indicator read by a Clinitek 200+, Ames, Elkhart, Indiana). The urine sediments from a single pooled sample/sex/dose group were microscopically evaluated. - Sacrifice and pathology:
- All animals were submitted for necropsy. Rodents submitted alive for necropsy were anesthetized by the inhalation of methoxyflurane vapors, blood/serum samples were obtained from the orbital sinus, their tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted by a veterinary pathologist assisted by a team of trained individuals.
GROSS PATHOLOGY: Yes
- Organs weights examined: The brain, liver, kidneys, heart, adrenals, testes, epididymides, thymus, spleen.
HISTOPATHOLOGY: Yes
- Number of animals: A complete histopathology was performed on the control and high dose animals. Livers were examined from the 100 and 500 mg/kg bw/day animals.
ORGANS AND TISSUES EXAMINED
Adrenals, aorta, auditory sebaceous glands*, bone (including joint)*, bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, duodenum, epididymides, oesophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/harderian glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.
* Tissues not examined histopathologically. - Statistics:
- All parameters examined statistically were first tested for equality of variance using Bartlett's test. If the results from Bartlett's test were significant, then the data for the parameter were subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. When Bartlett's test was satisfied, that form of the data was used. If none of the transformations or the raw data resulted in homogeneous variances, the data was reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis as described in the field “Any other information on materials and methods incl. tables”.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Significantly lower feed consumption (day 23-28) for males in the 1000 mg/kg bw/day test group.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Liver and kidney weight increased in all rats fed ≥ 500 mg/kg/day.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Centrilobular hepatocellular hypertrophy observed in females fed ≥ 500 mg/kg bw/day and males fed 1000 mg/kg bw/day.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
One female in the 100 mg/kg bw/day treatment group was observed with perineal soiling during the prestudy clinical observation as well as in weeks 1-3. This condition was no longer present at the time of the final clinical observation and was considered unrelated to treatment. There were no remarkable observations in the animals in the remaining dose groups.
BODY WEIGHT AND WEIGHT GAIN
There were no statistically identified differences in the male or female body weights of any treated group when compared to their respective controls. All male and female treated groups generally had body weight gains equivalent to or in excess of the control groups. At several time points, the 500 and 1000 mg/kg bw/day females had body weight gains that were statistically higher than the controls.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: The feed consumption for Days 23-28 calculated for the male 1000 mg/kg bw/day treated group was significantly lower than the control. There were no significant differences in the amount of feed consumption by any other treated group when compared to their respective controls.
- Time Weighted Average dosages: Due to variations in the percentage of test material in the test diets, the time weighted average (TWA) dosage was calculated. The TWA dosages of the test material which the test animals actually ingested were calculated based on the percent of the test material in the diet, body weights and feed consumption for each respective week of study. Rats at all dosages consumed levels of test material that were within 10 % of the targeted concentrations.
HAEMATOLOGY
There were no haematological changes considered to be treatment related. Small increases in red blood cell count (RBC) and haematocrit (HCT) were statistically identified for both sexes combined only at the middle dose of 100 mg/kg/day; but, the direction of this change, and the lack of a dose-response, indicate a random event unrelated to treatment.
CLINICAL CHEMISTRY
Statistically identified differences from control groups values occurred as follows. Alkaline phosphatase (ALP) was slightly increased in both sexes combined at doses ≥ 100 mg/kg bw/day. Blood glucose (GLUC) was decreased in a non-dose related manner in both sexes combined at doses ≥ 100 mg/kg bw/day. Total protein (TP) and albumin (ALB) were increased at 1000 mg/kg bw/day in both sexes combined. Cholesterol (CHOL) was increased in females at doses ≥100 mg/kg bw/day, and in males at only the 500 mg/kg bw/day dose. Serum phosphorus was decreased slightly in both sexes combined at 1000 mg/kg bw/day.
None of these differences were biologically or toxicologically significant. All were within the range of recent historical controls for this laboratory, with the exception of the CHOL value of 73 mg/dl for the 500 mg/kg bw/day male rats; the historical range for that value was 42-64 mg/dl. However, the male 1000 mg/kg bw/day group mean value for cholesterol was 57 mg/dl; indicating a random, non-treatment-related, biologically non-significant event in the 500 mg/kg bw/day males.
URINALYSIS
There was no effect of treatment on any urinalysis parameter.
ORGAN WEIGHTS
- Liver: Absolute and relative increases in liver weight were statistically identified in both sexes combined, and attributed to test material ingestion. In males fed 500 and 1000 mg/kg bw/day the increases in absolute liver weights were 17.9 % and 28.2 %, respectively. In females fed 500 and 1000 mg/kg bw/day the increases in absolute liver weight were 23.7 % and 24.9 %, respectively.
- Kidneys: The weights of kidneys were also increased. At 1000 mg/kg bw/day the absolute weights were statistically increased for both sexes combined; for males the increase was 16.8 %, for females 4.3 %. The relative kidney weight was also statistically identified as increased for males at 1000 mg/kg/day. At 500 mg/kg/day small increases in absolute kidney weight were statistically identified for both sexes combined (6.4 % for males and 6.7 % for females). Small differences in kidney weight, like those seen in the 1000 mg/kg bw/day females when combined with the males for statistical purposes, and in both sexes combined at 500 mg/kg bw/day, are questionable in their relation to treatment.
GROSS PATHOLOGY
There were no gross pathological findings related to treatment.
HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver: The histopathologic correlate of increased liver weight, centrilobular hepatocellular hypertrophy, was observed in all of the livers of both sexes at 1000 mg/kg bw/day and the livers of all of the females at 500 mg/kg bw/day.
Slight to moderate, focally-extensive, liver necrosis was observed in three rats: a female given 10 mg/kg bw/day, and two males in the 500 mg/kg bw/day group. These were significant lesions, but with no apparent relationship to ingestion of the test material; they appeared to be most consistent with a bacterial etiology. All were located in the right medial lobe, around the base of the fissure dividing the left and right medial lobes.
- Kidney: No histopathological correlate to the increases in kidney weights was detected.
- Other: The other lesions observed were generally typical spontaneous findings in rats of this age and stock, and were unrelated to treatment. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Based on liver and kidney weight increases in all rats fed ≥ 500 mg/kg/day, and centrilobular hepatocellular hypertrophy in females fed ≥ 500 mg/kg/day and males fed 1000 mg/kg/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: The observed changes (increased liver and kidney weights, and centrilobular hepatocellular hypertrophy) were considered to be adaptive.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the no-observed-effect-level (NOEL) was the targeted concentration of 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) was the highest targeted concentration 1000 mg/kg bw/day.
- Executive summary:
The subacute toxicity of the test material was assessed in a 4 week dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guideline OECD 407.
Five male and five female Fischer 344 rats per group were given test diets formulated to supply 0, 1, 10, 100, 500 or 1000 mg/kg bw/day for 4 weeks. Calculated actual dosages received, based on average body weight and feed consumption data were: males - 0, 1.0, 10.4, 101.4, 512.6, and 1029.1 mg/kg bw/day and females - 0, 1.1, 10.9, 105.1, 520.6, and 1055.6 mg/kg bw/day. Standard toxicologic parameters were evaluated.
Group body weights or body weight gains for both sexes were similar to control, or in excess of control values, throughout the study. Feed consumption for males and females were generally similar to controls over the four weeks of the study.
No treatment-related effects were observed in any of the haematology, clinical chemistry or urinalysis parameters examined.
Organ weight effects were limited to increased liver and kidney weights in both sexes given 500 or 1000 mg/kg bw/day. There were no gross pathological findings attributable to treatment. The histopathologic findings that were treatment-related were limited to centrilobular hepatocellular hypertrophy in the livers of both sexes ingesting 1000 mg/kg bw/day and the livers of all females given 500 mg/kg bw/day.
Based on the parameters evaluated in this four week study, the no-observed-effect-level (NOEL) for was the targeted concentration of 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) was the highest targeted concentration 1000 mg/kg bw/day.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 25 July 2001 - 22 January 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- There are no guidelines applicable to the duration and route of administration of this dog study.
Male and female Beagle dogs (two/sex/dose level) were fed diets formulated to contain the test material at concentrations of 0, 0.3, 0.9 or 3.0 % daily for 28 consecutive days. These concentrations corresponded to dose levels of approximately 102.7, 319.1, and 1034.9 mg/kg bw/day in males, and 90.8, 366.0, and 1143.5 mg/kg bw/day in females. Parameters evaluated included clinical appearance, ophthalmologic examinations, body weight, feed consumption, clinical chemistry (including prothrombin time), haematology, urinalysis, selected organ weights, gross and histopathologic examinations. - GLP compliance:
- yes
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately 6 months old.
- Housing: Individually, in pens approximately 1 meter in width, 2 meters in length, and 1.5 meters in height.
- Diet: Canine diet in meal form provided ad libitum.
- Water: Municipal water provided ad libitum.
- Acclimation period: Dogs were acclimated to the laboratory for eighteen days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24 °C
- Humidity (%): 53 – 70 %
- Air changes (per hr): 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: 06 August 2001 To: 04 September 2001 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared weekly.
- Mixing appropriate amounts with (Type of food): The high-dose diet was prepared by mixing test material with control feed to obtain the desired percent of test material in the diet. Mid- and low-dose diets were prepared by serially diluting the high-dose level diet with control feed. Actual test material intake was calculated based on diet concentrations, feed consumption, and body weight data collected throughout the study. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: Homogeneity of the low- and high-dose test material-feed mixtures was determined prior to the start of the study.
Stability: The stability of the test material in the feed was initiated prior to the start of the study on the low- and high-dose diets and was further determined approximately 15 days later.
- Concentration Verification: Analyses of low-dose, high-dose, and control diets were conducted during the study. The method for analysing the test material in feed was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Retainer Samples: Reference samples (one/dose level/mix) were retained and stored at ambient temperature in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- 28 consecutive days.
- Frequency of treatment:
- Daily.
- Remarks:
- Doses / Concentrations:
0, 0.3, 0.9 and 3.0 %
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
102.7, 319.1, and 1034.9 mg/kg bw/day (males)
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
90.8, 366.0, and 1143.5 mg/kg bw/day (females)
Basis:
nominal in diet - No. of animals per sex per dose:
- Two per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose level of 3 % represented the limit test dose as specified by several regulatory agencies for oral toxicity studies in non-rodents. The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL).
- Dose route rationale: Probable routes of human exposure to the test material would be via accidental ingestion during application or manufacture. Thus, administration of the test material to Beagle dogs via the dietary route represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day.
- Cage side observations (where possible): Skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Examinations were conducted on all animals pre-exposure and weekly throughout the study period.
- DOC observations: This examination included careful, hands-on evaluations of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behaviour.
BODY WEIGHT: Yes
- Time schedule for examinations: All dogs were weighed during the pre-exposure period, twice during the first week and weekly during the remainder of the study. Body weight gains were calculated throughout the duration of the study.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Feed consumption: Data was collected pre-exposure, twice during the first week of dosing, and weekly thereafter for all animals. Feeders were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) /No. of days in measurement cycle
- Test material intake (TMI): TMI was calculated using actual feed concentrations, body weights and feed consumption data in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Eyes were examined pre-exposure and during the week prior to necropsy using indirect ophthalmoscopy, and during necropsy using a moistened glass slide pressed to the cornea.
- Dose groups that were examined: All animals were examined.
- Method: Eyes were examined by a veterinarian. One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected pre-exposure and the day of necropsy via venipuncture of the jugular vein.
- Anaesthetic used for blood collection: Yes (sodium pentobarbital and exsanguination).
- Animals fasted: Yes
- How many animals: All animals
- Sample preparation:
> Complete blood count: Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and archived. Haematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
> Coagulation: Blood samples were collected in sodium citrate tubes, centrifuged and plasma was collected and assayed using an MLA Electra 700, Automatic Coagulation Timer.
- Parameters checked:
> Complete blood count: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, RBC indices (MCH, MCV and MCHC), and methaemoglobin concentration (only determined at study termination).
> Coagulation: Prothrombin time (PT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected pre-exposure and the day of necropsy via venipuncture of the jugular vein.
- Animals fasted: Yes
- How many animals: All animals
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN).
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all animals (via aspiration from the urinary bladder) during the scheduled necropsy.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: pH, bilirubin, glucose, proteins, ketones, blood, and urobilinogen. - Sacrifice and pathology:
- Fasted dogs were weighed, tranquilised by the subcutaneous administration of acepromazine (0.3 mg/kg), and subsequently euthanised by an intravenous overdose of sodium pentobarbital and exsanguination. Terminal body weights were recorded.
A veterinary pathologist assisted by a team of trained individuals conducted a complete necropsy on all animals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. Bone marrow smears were prepared from the proximal femur and stained with May-Grunwald stain, but were not examined. Transponders were removed and placed in jars with the tissues.
GROSS PATHOLOGY: Yes
- Dose groups examined: All animals
- Organ weights examined: The liver and kidneys were trimmed and weighed and organ to body weight ratios calculated.
HISTOPATHOLOGY: Yes
- Dose groups examined: All control and high-dose group animals.
- Tissues examined: Adrenals, aorta, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, cranial nerve - optic, colon, duodenum, epididymides, esophagus, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues - pharynx, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, urinary bladder, uterus and vagina. - Statistics:
- Descriptive statistics only (mean and standard deviations) were performed on urine specific gravity, and on feed consumption, body weight, organ weight, haematologic, and clinical chemistry parameters.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Mortality:
- no mortality observed
- Description (incidence):
- No treatment related effects.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: All dogs survived the dosing period.
- Detailed clinical observations (DCO's): There were no treatment-related alterations in DCO’s. All DCO’s were consistent with those expected in dogs. One low-dose male had an umbilical hernia prior to study start.
BODY WEIGHT AND WEIGHT GAIN
All study animals had body weight gains that were comparable to controls.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed consumption: All study animals had feed consumption values that were comparable to controls.
- Test material intake (TMI): Dose levels in mg/kg bw/day were calculated from diet concentrations (%), body weight (kg), and food consumption (g/day) data. The average intake for males given 0.3 %, 0.9 %, or 3.0 % test material was 102.7, 319.1, and 1034.9 mg/kg bw/day, respectively. The average intake for females given 0.3 %, 0.9 %, or 3.0 % was 90.8, 366.0, and 1143.5 mg/kg bw/day, respectively.
OPHTHALMOSCOPIC EXAMINATION
Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.
HAEMATOLOGY
> Coagulation: There were no treatment-related alterations in the prothrombin times of male and female dogs at any dose level.
> Haematology: There were no treatment-related alterations in any of the haematologic parameters for male dogs at any dose level.
Both females given the test material at 0.9 % and both females given 3.0 % had slightly lower values for haemoglobin concentration, haematocrit, and/or red blood cell count, relative to controls (see Table 1). However, these values were within or near historical control values of 28-day dietary toxicity dog studies recently conducted at this laboratory, the lower values did not occur in a dose-responsive manner, and there were no corresponding histopathologic alterations. In addition, the haematologic parameters of female control group animals were higher, or near the upper range of historical control values. Therefore, the slightly lower red blood cell parameters of female dogs were interpreted to represent normal biologic variability between animals, and were unrelated to treatment with the test material.
There were no treatment-related alterations in percent of methaemoglobin in the blood of male and female dogs at any dose level. Methaemoglobin values for all dose levels of males and females were slightly higher than concurrent controls. However, all animals had methaemoglobin levels within the normal range of 0.5 to 3.0 % for dogs (Harvey, 1989).
CLINICAL CHEMISTRY
There were no treatment-related alterations in any of the clinical chemistry parameters of male and female dogs at any dose level.
URINALYSIS
There were no treatment-related alterations in any of the urinalysis parameters of male and female dogs at any dose level.
ORGAN WEIGHTS
There were no treatment-related alterations in liver and kidney weights of male and female dogs at any dose level.
GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.
HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related histopathologic observations. All histopathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 3 other: %
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Based on no treatment-related effects observed in any of the study parameters examined up to the maximum dose level. 3.0 % is equivalent to dose levels of 1034.9 and 1143.5 mg/kg bw/day in males and females, respectively.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, there were no treatment-related effects in any of the above parameters. The no-observed-effect level (NOEL) for males and females was 3.0 % (1034.9/1143.5 mg/kg bw/day, in males and females respectively).
- Executive summary:
The subacute toxicity of the test material was assessed in a 28 week dietary toxicity study using Beagle dogs. The study was performed under GLP conditions and was performed in accordance with sound scientific principles.
Male and female Beagle dogs (two/sex/dose) were fed diets formulated to contain the test material at 0, 0.3, 0.9 or 3.0 %. These concentrations corresponded to dose levels of approximately 102.7, 319.1, and 1034.9 mg/kg bw/day in males, and 90.8, 366.0, and 1143.5 mg/kg bw/day in females. Parameters evaluated included clinical appearance, ophthalmologic examinations, body weight, feed consumption, clinical chemistry (including prothrombin time), haematology, urinalysis, selected organ weights, gross and histopathologic examinations.
Under the conditions of the test, there were no treatment-related effects in any of the above parameters. The no-observed-effect level (NOEL) for males and females was 3.0 % (1034.9/1143.5 mg/kg bw/day, respectively).
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 7 November 2001 - 12 September 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF Guidelines Subchronic Oral Toxicity Study
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Insecticide Law of India B.i. Sub-acute Toxicity Studies in Rats and Dogs for 90 Days
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 months.
- Housing: Individually one per pen, pens were approximately 1 meter in width, 2 meters in length, and 1.5 meters in height.
- Diet: Certified canine diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Male and female dogs were acclimated to the laboratory for 34 and 35 days, respectively.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 40 to 62 %
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: Males 21 November 2001, and females 22 November 2001 To: Males 20 February 2002, females 21 February 2001 and the recovery group on 22 March 2002. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: High-dose diets were prepared weekly.
- Mixing appropriate amounts with: To produce the high dose diet, the test material was mixed with control lab diet to obtain the desired percent of test material in the diet. Mid-dose diets were prepared by diluting the high-dose level diet with control lab diet, and the low-dose diets were prepared by diluting the mid-dose level diet with control lab diet. Concentration of the test material in the feed was held constant throughout the dosing period. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low- and high-dose diets was determined pre-exposure and near the middle and at the end of the study.
- Stability: Stability determination was initiated prior to the study start at concentrations of 0.003 % and 3 % and further determined 12 and 27 days after initial analysis.
- Concentration verification: Analyses of the test material in the diet at all levels, plus control were determined pre- exposure, near the middle, and at the end of the study. The method for analysing test material in feed was a solvent extraction method followed by analysis using high performance liquid chromatography (HPLC) with mass spectrometric (MS) detection using internal standard quantitation.
- Retainer samples: Reference samples (one/dose/mix) were retained and stored at ambient temperature in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- 90 consecutive days.
- Frequency of treatment:
- Daily.
- Remarks:
- Doses / Concentrations:
0, 0.003, 0.3 or 3.0 %
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 0.913, 115, and 1040 mg/kg bw/day (males)
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 1.06, 113, and 1150 mg/kg bw/day (females)
Basis:
nominal in diet - No. of animals per sex per dose:
- Four per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose level of 3 % was expected to be well tolerated by dogs, based on the lack of treatment-related effects at this dose level during the four-week dog study. The remaining dose levels were expected to provide dose-response data for any treatment-related effect (s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL).
- Dosing route rational: Probable routes of human exposure to the test material would be via accidental ingestion during field application or manufacture. Thus, administration of the test material to Beagle dogs via the dietary route represents an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program. Littermates were not included in the same treatment groups.
- Post-exposure recovery period in satellite groups: Additional “recovery” groups of four males and four females per dose level were given diets containing the test material at 0 or 3 % for 90 days, and then were maintained on control diet for 28 days. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day
- Cage side observations checked (where possible): skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour and the availability of feed and water. In addition, animals were examined twice daily for moribundity or mortality.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted pre-exposure and weekly on all dogs throughout the study. The DCO was conducted at approximately the same time each examination day according to an established format.
- DCO included: The examination included cage-side, hands-on and open-field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: All dogs were weighed during the pre-exposure period and weekly during the study. Body weight gains were also calculated.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected pre-exposure and least during the study. Feeders were weighed at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) /(No. of days in measurement cycle)
- Test material intake (TMI): TMI was calculated using actual feed concentrations, body weights and feed consumption data in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Eyes were examined by a veterinarian pre-exposure and during the week prior to necropsy using indirect ophthalmoscopy. Recovery phase group animals were examined pre-exposure.
- Dose groups that were examined: All animals were examined.
- Method: One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during the necropsy using a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected via venipuncture of the jugular vein from all animals pre-exposure, midway through the treatment period (main study animals only), during week 13, and week 17 (haematology parameters for recovery study dogs only).
- Anaesthetic used for blood collection: Yes (acepromazine, 0.3 mg/kg).
- Animals fasted: Yes
- How many animals: All animals
- Sample preparation (complete blood count): Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright’s stain and examined microscopically by a veterinary pathologist. Haematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
- Sample preparation (coagulation): Blood samples were collected in sodium citrate tubes, centrifuged and plasma was collected and assayed using an MLA Electra 700, Automatic Coagulation Timer.
- Parameters checked (complete blood count): Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, RBC indices (MCH, MCV and MCHC), and methaemoglobin concentration.
- Parameters checked (coagulation): Prothrombin time (PT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected via venipuncture of the jugular vein from all animals pre-exposure, midway through the treatment period (main study animals only), during week 13, and week 17 (hematology parameters for recovery study dogs only).
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGT).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN)
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from dosing-phase animals for timed urine volume determination prior to, midway through the treatment period, and near the end of the study. Recovery-phase dogs also had urine collected pre-exposure, and during weeks 13 and 17 (females only at weeks 13 and 17). Week 17 urine samples were collected by aspiration of the urinary bladder at necropsy.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Colour, appearance, specific gravity* (refractometer) and urine volume*.
Semiquantitative analyses of: pH, bilirubin, glucose, proteins, ketones, blood, urobilinogen, and characterization of the microsediment*
* = dosing phase animals only. - Sacrifice and pathology:
- Fasted dogs were weighed, tranquilised by the subcutaneous administration of acepromazine (0.3 mg/kg), and subsequently euthanised by an intravenous overdose of sodium pentobarbital and exsanguination.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle. Bone marrow smears were prepared from the proximal femur and stained with May-Grunwald stain and examined by a veterinary pathologist.
GROSS PATHOLOGY: Yes
- Organ weights: brains, livers with gallbladders (incised), kidneys, hearts, testis, epididymides, ovaries, uteri, thymuses, spleens, adrenal glands and thyroids with parathyroid glands. For the recovery phase group animals, the liver and kidneys were trimmed and weighed at necropsy.
HISTOPATHOLOGY: Yes
- Tissues examined: Adrenals, aorta, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, cranial nerve – optic, colon, duodenum, epididymides, esophagus, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues - pharynx, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, urinary bladder, uterus and vagina. - Statistics:
- Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption is addressed below) were first tested for equality of variance using Bartlett's test (Winer, 1971). If the results from Bartlett's test were significant at alpha = 0.01, then the data for the parameter was subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data was reviewed and an appropriate form of the data selected. The selected form of the data was then subjected to the appropriate parametric analysis as described below "Any other information on materials and methods incl. tables".
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Mortality:
- no mortality observed
- Description (incidence):
- No treatment related effects.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment related effects observed in male and females dosed at concentration ≥ 0.3 %, see 'Details on results' below.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Higher relative mean liver and kidney weights (males and females give 3.0 %); absolute mean liver and kidney weights were higher than the controls, but not statistically identified (males and females give 3.0 %).
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Erythroid cell hyperplasia of the bone marrow (males and females given ≥ 0.3 %)
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: All dogs survived the 13-week dosing period and the 4-week recovery period.
- DCO: There were no treatment-related DCOs. The most common observation was soft faeces, which occurred sporadically in males and females from controls and all treatment groups. All other DCOs were consistent with those expected in Beagle dogs of this age.
BODY WEIGHT AND WEIGHT GAIN
There were no statistically identified changes in body weights of males and females at any dose level over the course of the study. At the end of the 13 week test material administration period, low-dose males gained 19 % less weight than controls, the mid-dose males gained 4 % less weight than controls, while the high-dose males gained 16 % less weight than controls. The low-dose females gained 13 % less weight than controls, the mid-dose females gained 31 % less weight than controls, while the high-dose females gained 6 % less weight than controls. These differences were not dose related and were considered representative of the normal weight range for dogs of this age.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: There were no treatment-related changes in feed consumption at any dose level.
- Test Material Intake: The average test material intake for males given diets containing the test material at 0.003, 0.3, or 3.0 % was 0.931, 115, or 1040 mg/kg bw/day, respectively. The average intake for females given diets containing the test material at 0.003, 0.3, or 3.0 % was 1.06, 113, or 1150 mg/kg bw/day, respectively. These values corresponded well with the expected dose levels (1, 100, and 1000 mg/kg bw/day) for these dietary concentrations.
OPHTHALMOSCOPIC EXAMINATION
Examinations performed on all animals prior to the study and at termination revealed no treatment-related findings. A hypo-pigmented fundus was noted in multiple animals, both pre-exposure and terminal, and was considered a normal variation in this strain of dog.
HAEMATOLOGY
- Coagulation: There were no treatment-related alterations in the prothrombin times of male and female dogs at any dose level over the duration of the study.
- Haematology: The mean red blood cell count of mid-dose (0.3 %) and high-dose (3.0 %) males and females combined over the duration of the study was statistically identified as lower than controls. However, only the males from these dose levels had mean red blood cell counts that were lower than pre-exposure values. This alteration in mid- and high-dose males was interpreted to be treatment related. There was complete recovery of the red blood cell counts for high-dose dogs following the 28-day recovery period.
The mean platelet count of mid-dose (0.3 %) and high-dose (3.0 %) males and females combined over the duration of the study was statistically identified as higher than controls. This alteration was interpreted not to be treatment related, because the high-dose male and female platelet counts at 13-weeks were lower than their respective pre-exposure values. The mean platelet count of high-dose dogs was still statistically identified as higher than controls following the 28-day recovery period, but they were comparable to their respective pre-exposure values.
Males given diet containing the test material at 0.3 % or 3.0 % had treatment-related decreases in mean haemoglobin concentration and haematocrit, however, these decreases were not statistically identified. There was complete recovery of these parameters for high-dose (3.0 %) males following the 28-day recovery period.
Males and females given diet containing the test material at 0.3 % or 3.0 % had treatment-related increases in mean corpuscular volume. There was complete recovery of this parameter for high-dose (3.0 %) males, and partial recovery of this parameter for high-dose (3.0 %) females, following the 28-day recovery period.
Males given diet containing the test material at 3.0 % had a treatment-related increase in percent neutrophils (NEUT) and a treatment-related decrease in percent lymphocytes (LYMP) of the peripheral blood, relative to controls. There was complete recovery of these parameters for high-dose (3.0 %) males following the 28-day recovery period.
There were no treatment-related alterations in percent of methaemoglobin in the blood of male and female dogs at any dose level.
Microscopic evaluation of blood smears taken near the end of the dosing phase revealed a very slight increase in polychromasia for one female given diet containing the test material at 0.3 %, and three males and one female given 3.0 %. This alteration was interpreted to treatment related and consistent with a regenerative response to the altered erythrocytic parameters of dogs from these dose levels.
CLINICAL CHEMISTRY
There were no treatment-related alterations in any of the clinical chemistry parameters for male or female dogs at any dose level during the study.
URINALYSIS
There were no treatment-related alterations in any of the urinalysis parameters for male and female dogs at any dose level during the study. Three females given diet containing the test material at 3.0 % had higher urine pH than any of the control group females at the end of the 13-week dosing period. However, the urine pH of all recovery phase females given 3.0 % for 13-weeks was comparable to recovery phase group controls. In addition, after 4 weeks of recovery, the urine pH of all females given 3.0 % was comparable to recovery phase group controls. The higher urine pH of high-dose (3.0 %) females from the main study may have been related to the length of time the urine sat in metabolism cage containers prior to analysis. Urine pH increases with time of exposure to air (Duncan et al., 1994).
ORGAN WEIGHTS
Animals given diet containing the test material at 3.0 % had treatment-related statistically identified higher relative mean liver and kidney weights, averaged across both sexes, relative to controls. Absolute mean liver and kidney weights of high-dose (3.0 %) males and females were also higher than controls, but not statistically identified. There were no corresponding microscopic alterations of the liver or kidneys.
There was complete recovery of the liver and kidney weight alterations following the 4-week recovery period.
GROSS PATHOLOGY
All gross pathologic observations were considered to be spontaneous alterations, unassociated with ingestion of the test material.
HISTOPATHOLOGY: NON-NEOPLASTIC
Male and female dogs given diet containing the test material at 0.3 or 3.0 % had treatment related erythroid cell hyperplasia of the bone marrow. The erythroid cell hyperplasia was indicative of a regenerative response to the altered erythrocytic parameters of dogs from these dose levels.
There was partial recovery of the erythroid cell hyperplasia of the bone marrow following the 4-week recovery period.
All other histopathologic observations were considered - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 0.003 other: %
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the no-observed-effect level (NOEL) for male and female dogs administered the test material in the diet for 90 days was 0.003 % (approximately 1 mg/kg/day).
- Executive summary:
The subchronic toxicity of the test material was assessed in a 90 day dietary toxicity study using Beagle dogs. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 409, EPA OPPTS 870.3150, EU Method B.27, JMAFF guidelines subchronic oral toxicity study, and Insecticide Law of India B.i. sub-acute toxicity studies in rats and dogs for 90 days.
Four males and four female Beagle dogs per dose level were fed diets formulated to supply 0, 0.003, 0.3 or 3.0% test material for 90 days. These concentrations corresponded to dose levels of approximately 0, 0.913, 115, and 1040 mg/kg bw/day in males, and 0, 1.06, 113, and 1150 mg/kg bw/day in females. Additional “recovery” groups of four males and four females per dose level were given diets containing the test material at 0 or 3 % for 90 days, and then were maintained on control diet for 28 days. Parameters evaluated included daily observations, detailed clinical observations, ophthalmic examinations, body weights, feed consumption, clinical pathology, organ weights, and gross and histopathologic examinations.
Following 13 weeks of dosing, males given diets containing the test material at 0.3 or 3.0 % had treatment related decreases in red blood cell count, haematocrit and haemoglobin concentration. In addition, the mean corpuscular volume of males and females given 0.3 or 3.0 % was increased.
Microscopic alterations that were secondary and compensatory to these hematologic effects that consisted of very slight or slight erythroid cell hyperplasia of the bone marrow and very slight polychromasia of red blood cells occurred in male and female dogs given 0.3 or 3.0 %. Following the four week recovery period, there was complete recovery of the alterations in red blood cell count, and hematocrit and hemoglobin concentration, and partial recovery of the bone marrow hyperplasia and increased mean corpuscular volume. Additional treatment related effects consisted of increased liver and kidney weights of males and females dosed at 3.0 %. There was complete recovery of the liver and kidney weight alterations following the four week recovery period.
The no-observed-effect level (NOEL) for male and female dogs administered the test material in the diet for 90 days was 0.003 % (approximately 1 mg/kg/day).
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 8 July 2002 - 5 March 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.4100 (Chronic Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 452 (Chronic Toxicity Studies)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.30 (Chronic Toxicity Studies)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately 7 months.
- Housing: Individually one per pen. Pens were approximately 1 meter in width, 2 meters in length, and 1.5 meters in height.
- Diet: Certified canine diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Dogs were acclimated to the laboratory for at least 14 days prior to the to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24 °C
- Humidity (%):44.5 to 70 %
- Air changes (per hr): approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: 11 July 2002 (males) and 12 July 2002 (females) To: 14 July 2003 (males) and 15 July 2003 (females). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: Diets were prepared at least every two weeks.
- Mixing appropriate amounts with: Diets were prepared by mixing the test material with control feed to produce the high-dose diet. Mid-dose diets were prepared by diluting the high-dose level diet with control lab diet, and the low-dose diets were prepared by diluting the mid-dose level diet with control lab diet. Concentration of the test material in the feed was held constant throughout the study. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low- and high-dose diets were determined prior to the study start, and during months 4, 9, and 12 of the study.
- Stability: Data from a 90-day toxicity study demonstrated that the test material was stable in canine lab diet for at least 21 days at concentrations of 0.003 and 3.0 % (Stebbins, et al., 2003), which brackets the doses used in this study. No additional stability analyses were conducted as part of this study.
- Concentration Verification: Analyses of test material in the diet at all levels was conducted prior to the start of the study, and during months 4, 9, and 12 of the study. The method for analysing the test material in feed was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Retainer Samples: Reference samples (one/sex/dose/mix plus control) were retained and stored at ambient temperature in sealed vials in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- One year.
- Frequency of treatment:
- Daily.
- Remarks:
- Doses / Concentrations:
0, 0.003, 0.03, or 0.225 %
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 0.74, 9.3, and 69 mg/kg bw/day (males)
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 0.94, 8.7, and 70 mg/kg bw/day (females)
Basis:
nominal in diet - No. of animals per sex per dose:
- Four per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose of 0.225 % was chosen based on the results of the 90-day study and was expected to produce treatment-related effects, including changes in erythrocytic parameters and an increase in liver weights (Stebbins, et al. 2003). The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL).
- Route selection rationale: Possible routes of human exposure to the test material would be via accidental ingestion during application or manufacture. Thus, administration of the test material to Beagle dogs via the dietary route represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once each day. In addition, animals were examined twice daily for moribundity, mortality, and the availability of feed and water.
- Cage side observations included (where possible): skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), and animal behaviour.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted at pre-exposure and weekly throughout the study. The DCO were conducted on all animals, at approximately the same time each examination day according to an established format.
- DCO observations included: The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: The dogs were weighed during the pre-exposure period, weekly during the first 13 weeks, and at least once every four weeks thereafter. Body weight gains were calculated throughout the duration of the study.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected pre-exposure, weekly during the first 13 weeks, and at least every four weeks thereafter for all animals. Feed crocks were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed crocks - final weight of feed crocks) / [(No. of days in measurement cycle)(No. of animals per run)]
- Test material intake (TMI): TMI was calculated using diet concentrations, mean body weights, and feed consumption values in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to termination using indirect ophthalmoscopy.
- Dose groups that were examined: All dose groups.
- Method: One drop of 0.5% tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected via venipuncture of the jugular vein from all animals pre-exposure, during weeks 12 and 26, and the morning of necropsy.
- Anaesthetic used for blood collection: Yes (acepromazine, 0.1 - 0.3 mg/kg)
- Animals fasted: Yes
- How many animals: All animals.
- Haematology sample preparation: Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and examined microscopically by a veterinary pathologist. Haematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
- Coagulation sample preparation: Blood samples for were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000.
- Haematology parameters checked: Haematocrit (HCT), haemoglobin (HGB) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, reticulocyte count, and RBC indices - mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular, haemoglobin concentration (MCHC).
- Coagulation parameters checked: Prothrombin time (PT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected via venipuncture of the jugular vein from all animals pre-exposure, during weeks 12 and 26, and the morning of necropsy.
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transpeptidase (GGT).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREA), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLUC), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN).
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected for timed urine volume determination pre- exposure, during weeks 13 and 27, and the morning of necropsy for both sexes. During collection periods, dogs were placed in metabolism cages fitted with urine collection vessels at approximately 3 PM until approximately 7 AM the following morning. Water, but no feed, was supplied to the animals during the 16 hour collection periods.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
> Assays: Colour, appearance, specific gravity (refractometer) and urine volume.
> Semiquantitative analyses of: pH, bilirubin, glucose, proteins, ketones, blood, urobilinogen, and characterisation of the microsediment. - Sacrifice and pathology:
- Fasted dogs were weighed, tranquilised by the subcutaneous administration of acepromazine (0.1-0.3 mg/kg), and subsequently euthanised by an intravenous overdose of sodium pentobarbital and exsanguination.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. Bone marrow smears were prepared from the proximal femur and stained with May-Grunwald stain and examined by a veterinary pathologist.
GROSS PATHOLOGY: Yes
- Organ weights: brain, liver with gallbladder (incised), kidneys, heart, testes, ovaries, epididymides, uterus, spleen, adrenal glands, and thyroid with parathyroid glands.
HISTOPATHOLOGY: Yes
- Tissues examined: Adrenals, aorta, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, cranial nerve – optic, colon, duodenum, epididymides, esophagus, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues - pharynx, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, urinary bladder, uterus and vagina. - Statistics:
- Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption was addressed below) were first tested for equality of variance using Bartlett's test (Winer, 1971). If the results from Bartlett's test were significant at alpha = 0.01, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data were reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis as described below (in the field ‘Any other information on materials and methods incl. tables’).
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased incidence of soft faeces (males given 0.225 %).
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Increased incidence of soft faeces (males given 0.225 %).
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Marginally higher mean corpuscular volumes (males given 0.225 %); higher mean reticulocyte counts (males and females given 0.225 %); higher mean platelet count (males and females given 0.225 %); very slight polychromasia (males and females given 0.225 %).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Higher alkaline phosphate activity (females given 0.225 %).
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Higher relative liver weights (males given 0.03 and 0.225 %); higher absolute liver weights (males given 0.225 %).
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Erythroid cell hyperplasia of the bone marrow (males given 0.03 & 0.225 %, one female given 0.225 %); increase incidence/degree of mucosal lymphoid hyperplasia in the fundus and/or pylorus of the stomach (males given 0.03 & 0.225 %, females given 0.225 %)
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: All dogs survived the one-year test period.
- Detailed Clinical Observations: The only treatment-related clinical observation was an increased incidence of soft faeces in males given the test material at 0.225 %. Isolated occurrences of reduced or no faecal output were noted in one or two animals from all dose groups, but not in a dose-responsive manner. Various incidental observations determined not to be related to treatment consisted of: palpable masses (interdigital cysts or focal dermal inflammation), diffuse mammary enlargement, mucoid feces, excessive hair loss, papule/pustules of the skin, reddened skin and discoloured fur.
BODY WEIGHT AND WEIGHT GAIN
There were no statistically identified changes in body weights of males and females at any dose level over the course of the study. However, at study termination, males given the test material at 0.03 % had lower mean body weight (15.3 %) and lower mean body weight gain (41.2 %), relative to male controls. These differences were not dose related and were considered representative of the normal weight range for dogs of this age. The body weights and body weight gains of all other male and female treatment groups were comparable to controls.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: There were no treatment-related changes in feed consumption at any dose level. The feed consumption of all male and female treatment groups were comparable to controls.
- Test Material Intake: The average test material intake for animals fed diets containing the test material at 0, 0.003, 0.03 or 0.225 % was 0, 0.74, 9.3, and 69 mg/kg bw/day for males and 0, 0.94, 8.7, and 70 mg/kg bw/day for females, respectively.
OPHTHALMOSCOPIC EXAMINATION
Examinations performed on all animals prior to the study and at termination revealed no treatment-related findings. Hypo-pigmented fundus (bilateral) was observed in one control and one mid-dose male and one mid-dose female. This observation is considered a normal variation in this strain of dog.
HAEMATOLOGY
- Coagulation: There were no treatment-related alterations in the prothrombin times of male and female dogs at any dose level during the study.
- Haematology: There were no statistically identified alterations in the red blood cell count, reticulocyte count, haemoglobin concentration, haematocrit, or red blood cell indices of males or females at any dose level. However, males given the test material at 0.225 % had marginally higher mean corpuscular volumes, and males and females given 0.225 % had higher mean reticulocyte counts over the duration of the study, relative to controls. These haematologic alterations were interpreted to be treatment related and were consistent with the histopathologic finding of erythroid hyperplasia of the bone marrow in males and females given 0.225 %. Males given 0.003 or 0.03 %, and females given 0.03 % had slightly higher mean reticulocyte counts at the 6 month interval. However, these values were interpreted to be unrelated to treatment because the reticulocyte counts of the same dose groups were comparable to controls at the 3 month and 12 month intervals.
The mean platelet count of high-dose (0.225 %) males and females combined over the duration of the study was statistically identified as higher than controls. This alteration was interpreted to be treatment related because the platelet counts for high-dose males and females were consistently higher than concurrent and historical controls, and higher than pre-exposure values, at the 3, 6, and 12- month intervals. The mean platelet counts of mid-dose (0.03 %) males and females combined over the duration of the study were statistically identified as different than controls. The changes in platelet counts of mid-dose animals were interpreted to be unrelated to treatment, because the platelet counts of mid-dose males were lower than controls at all sampling intervals, and the platelet counts of mid-dose females were within or near to the historical control values at all sampling intervals.
Microscopic evaluation of blood smears revealed an increase in the incidence of very slight polychromasia of males and females given the test material at 0.225 % at the various sampling intervals. Very slight polychromasia was present in 1 of 4 males and 1 of 4 females given 0.225 % at 3 months, 2 of 4 males and 2 of 4 females given 0.225 % at 6 months, and 3 of 4 males and 2 of 4 females given 0.225 % at 12 months. This alteration was interpreted to be treatment related and consistent with the higher mean corpuscular volumes and higher reticulocyte counts of dogs from this dose level.
CLINICAL CHEMISTRY
The mean alkaline phosphatase activity of high-dose (0.225 %) males and females combined over the duration of the study was statistically identified as higher than controls. However, only females given the test material at 0.225 % had an elevated alkaline phosphatase activity that was interpreted to be treatment related. These elevated levels occurred at the 3 and 12 month sampling intervals, with individual high-dose female dogs 02A3902 and 02A3904 having the highest alkaline phosphatase activities throughout the 12 month exposure period. There was no explanation for the normal alkaline phosphatase activity in female dogs given 0.225 % at the 6 month sampling interval.
There was no histopathologic correlate to the higher alkaline phosphatase activities.
The mean chloride concentration of high-dose (0.225 %) males and females combined over the duration of the study was statistically identified as higher than controls. The chloride concentrations of high-dose males and females were within or very near the historical control values of Beagle dog dietary studies recently conducted at this laboratory. Therefore, the minimally increased chloride concentrations were interpreted to be unrelated to treatment, non-adverse, and reflective of normal biologic variation of this parameter.
The mean total bilirubin concentration of high-dose (0.225 %) males and females combined over the duration of the study was statistically identified as higher than controls. This alteration was interpreted not to be treatment-related because the bilirubin values were of minimal variation from concurrent controls, and were within published reference values of 0-3 mg/dl for dogs (Duncan et al., 1994).
URINALYSIS
The mean urine specific gravity of mid-dose (0.03 %) males and females combined over the duration of the study was statistically identified as different than controls. At the 3 month sampling interval, the specific gravity of mid-dose males was higher than controls, whereas the specific gravity of mid-dose females was lower than controls. At the 6 month and 12 month sampling intervals, the specific gravity of male and female mid-dose animals was lower than controls. These alterations were interpreted to be unrelated to treatment because of the variability of values at the 3 month sampling interval, and the lack of any statistically identified changes in urine specific gravity of high-dose animals.
ORGAN WEIGHTS
Animals given the test material 0.03 % or 0.225 % had statistically identified higher relative liver weights analysed across both sexes. The mean relative liver weights for males and females given diets containing the test material at 0.225 % were 29.7% and 12.3% higher than controls, respectively. The mean relative liver weights for males and females given 0.03 % were 27.5 % and 8.1 % higher than controls, respectively. Additionally, animals given 0.225 % had statistically identified higher absolute liver weights averaged across both sexes. The elevated relative and/or absolute liver weights in males given 0.03 % or 0.225 % were interpreted to be treatment related. However, the elevated relative liver weights of females given 0.03 or 0.225% test material were interpreted to not be treatment related because the relative weights were within historical controls values, and the lack of a dose response. The higher absolute liver weight of females given 0.225 % was interpreted to be reflective of the higher body weights of these animals, and not a treatment related alteration. There was no histopathologic correlate to the higher liver weights in either males or females.
Males given 0.03 or 0.225 % and females given 0.003, 0.03 or 0.225 % had higher absolute and relative adrenal weights compared to the concurrent and historical control values. Only the absolute adrenal weights of males and females given 0.225 % were statistically identified as different from the concurrent controls, when analysed using a two-way ANOVA with the factors of sex and dose. Treatment related histologic effects were not noted in the adrenals of either males or females given the test material at 0.225 %.
Examination of the male data indicated quite a degree of variability between individual male absolute and relative adrenal weights in control and treated animals.
Similar variability was also evident in the individual male historical control data, with individual absolute weights ranging from 0.991 – 1.767 g and the relative weights ranging from 0.009 to 0.014 g/100 g bw.
Adrenal weight differences of females given the test material at 0.225 % appeared to be due to 1) much higher absolute and relative adrenal weights of two females (3901 and 3902) of comparable body weight to the controls and 2) higher absolute adrenal weights and significantly higher body weights of the remaining two females (3903 and 3904), compared to the controls. Historical control adrenal weight data for females was less useful than the data from the males. This was due to the much lower body and adrenal weights of the historical controls than the concurrent controls from this study. However, the data clearly demonstrates the variability in relative adrenal weights of control females, with a range of 0.010 to 0.018 g/100 g bw.
In addition, significant variability in adrenal weights can be seen at the lowest dose level (0.003 %) with animal 3893 having a high relative adrenal weight (0.022g/100 g) compared to other animals in this dose level or the next higher dose level (0.03 %). Based on a weight of evidence approach, the minimal differences in adrenal weights of males and females given the test material at 0.225 % were interpreted not to be treatment related because of 1) the variability in individual animal adrenal weights between and within dose levels, 2) the lack of treatment-related histologic findings in the adrenal gland and 3) the fact that the adrenal was not identified as a target organ based on histologic findings or organ weight alterations in a 90-day study (Stebbins, et al., 2003) at higher dose levels.
GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered spontaneous alterations, unassociated with ingestion of the test material.
HISTOPATHOLOGY: NON-NEOPLASTIC
All males given diets containing the test material at 0.03 % or 0.225 % and one female given 0.225 % had treatment-related erythroid cell hyperplasia of the bone marrow. The erythroid cell hyperplasia was indicative of a regenerative response, and was consistent with the polychromasia and increases in mean corpuscular volumes and reticulocyte counts of dogs given 0.225 %.
Males given diets formulated at 0.03 % or 0.225 %, and females given 0.225 %, had a treatment-related increase in the incidence or degree of mucosal lymphoid hyperplasia in the fundus and/or pylorus of the stomach. This finding was a minor exacerbation of gastric lymphoid hyperplasia which is commonly seen in dogs. The incidence and severity of gastric lymphoid hyperplasia in males given 0.003 % and females given 0.003 % or 0.03 % were interpreted to be comparable to animals from the control group. There was no degeneration or necrosis of the gastric mucosa in any of the treated animals. Some animals from the controls and all treatment groups had inflammation of the gastric mucosa, however, there was no consistent correlation between the inflammation and the incidence of mucosal lymphoid hyperplasia.
All other histopathologic observations were considered to be spontaneous alterations, unassociated with the exposure to the test material. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 0.003 other: %
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: see 'Remark'
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 0.03 other: %
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: see 'Remark'
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the no-observed-effect level (NOEL) following one year of dietary exposure to the test material was 0.003 %, equivalent to 0.74 mg/kg bw/day for males, and 0.03 %, equivalent to 8.7 mg/kg bw/day for females.
- Executive summary:
The chronic toxicity of the test material was assessed in a 1 year dietary toxicity study using Beagle dogs. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 452, EPA OPPTS 870.4100 and EU Method B.30.
Male and female Beagle dogs (four/sex/dose) were fed diets formulated to provide the test material at concentration of 0, 0.003, 0.03, or 0.225 %. These concentrations corresponded to approximately 0, 0.74, 9.3, and 69 mg/kg bw/day in males, and 0, 0.94, 8.7, and 70 mg/kg bw/day in females. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, urinalysis, selected organ weights, and gross and histopathologic examinations.
Treatment-related effects were noted in red blood cell parameters of high-dose (0.225 %) animals. These effects consisted of higher mean corpuscular volumes in high-dose males, and higher reticulocyte counts with polychromasia of red blood cells in high-dose males and females. All males given the test material formulated at 0.03 % or 0.225 % and one female given 0.225 % had treatment-related erythroid cell hyperplasia of the bone marrow. The erythroid cell hyperplasia was indicative of a regenerative response, and was consistent with the polychromasia and increases in mean corpuscular volumes and reticulocyte counts of dogs from the high-dose group. The only other treatment-related haematologic effect was higher platelet counts in high-dose males and females.
The only treatment-related alteration in clinical chemistry parameters consisted of a higher alkaline phosphatase activity in high-dose females. High-dose males had higher absolute liver weights, and males given diets formulated at 0.03 % or 0.225 % had higher relative liver weights that were interpreted to be treatment related. There were no histopathologic correlates to the clinical chemistry or liver weight alterations. Males given diets formulated at 0.03 % or 0.225 %, and females given 0.225 %, had a treatment-related increase in the incidence and degree of mucosal lymphoid hyperplasia in the fundus and/or pylorus of the stomach.
The no-observed-effect level (NOEL) following one year of dietary exposure to the test material was 0.003 %, equivalent to 0.74 mg/kg bw/day for males, and 0.03 %, equivalent to 8.7 mg/kg bw/day for females.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 24 January 2001 - 12 June 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: approximately 7 weeks old.
- Housing: Individually, in stainless steel cages.
- Diet: Rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for seven days prior to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 22.0 °C
- Humidity (%): 45.3 - 60.4 %
- Air changes (per hr): Approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle, lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: 30 January 2001 To: 27 February 2001 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes and diets were mixed periodically and dietary concentrations adjusted based upon the most recent body weight and feed consumption data. Initial concentrations of test material in all diets were calculated from historical body weights and feed consumption data. All female diets fed during week two were calculated using historical feed consumption values and current bodyweight data due to excessive scratching of the feed. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low-dose female and the high-dose male test material-feed mixtures were determined prior to the start of the study.
- Stability: The stability of the test material in the vehicle was determined prior to start of treatment in a concurrent study. The stability of the test material in diets ranging in concentration from 0.005 % to 5 % was determined over at least a ten-day period.
- Concentration verification: Analyses of the premix, control, and all prepared diets were conducted prior to the study start. The method for analysing the test material in feed was a solvent extraction method followed by analysis using liquid chromatography (LC) with mass spectroscopy (MS) detection and internal as well as external standards.
- Retainer samples: Reference samples (one/sex/dose/mix) were retained and stored in sealed vials in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- 28 consecutive days
- Frequency of treatment:
- Daily
- Remarks:
- Doses / Concentrations:
0, 10, 100, 500, or 1000 mg/kg bw/day
Basis:
nominal in diet - Remarks:
- Doses / Concentrations:
0, 10.8, 110, 538, and 1060 mg/kg bw/day
Basis:
other: actual test material intake (males) - Remarks:
- Doses / Concentrations:
0, 11.2, 113, 504, and 1140 mg/kg bw/day
Basis:
other: actual test material intake (females) - No. of animals per sex per dose:
- Five per sex per dose.
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose level of 1000 mg/kg bw/day represented the limit test, as specified by several regulatory agencies for 28-day studies. The remaining dose levels were expected to provide dose-response data for any treatment-related effect(s) observed in the high-dose group and to ensure definition of a no-observed-effect level (NOEL) based on previous toxicity data.
- Previous toxicity data: The acute oral and dermal LD50 in Fischer 344 rats and New Zealand White rabbits were > 5000 mg/kg. The application of the test material moistened with an aqueous solution of methylcellulose showed no signs of irritation in an acute dermal irritation study using New Zealand White rabbits. In addition, the test material caused only slight to moderate eye irritation in New Zealand White rabbits which were resolved within 48 hours of treatment.
- Dose route rationale: Probable routes of human exposure to the test material would be via ingestion of foodstuffs that may contain low levels of residues or from accidental ingestion during field application or manufacture. Thus, administration of the test material to mice via the diet represents an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.
- Cage side observations: Skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed Clinical Observations (DCO) were conducted pre-exposure and weekly throughout the study. The DCO was conducted on all animals, at approximately the same time each examination day.
- DCO observations: The examination included cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: All mice were weighed during the pre-exposure period, twice during the first week and weekly during the remainder of the study.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected twice during the first week of dosing and weekly thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / [(No. of days in measurement cycle)(No. of animals per cage)]
- Test material intake (TMI): Test material intake (TMI) was calculated using actual feed concentrations, body weights and feed consumption data using the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Eyes were examined pre-exposure and prior to termination.
- Dose groups that were examined: The eyes of all animals were examined.
- Method: Eyes were examined by a veterinarian using indirect ophthalmoscopy. One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus at the scheduled necropsy.
- Anaesthetic used for blood collection: Yes (Carbon Dioxide)
- Animals fasted: No
- How many animals: All animals
- Sample preparation: Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and archived. Haematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
- Parameters checked: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential (WBC) count, and red indices (MCH, MCV & MCHC).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus at the scheduled necropsy.
- Animals fasted: No
- How many animals: All animals
- Sample preparation: Blood samples were collected in appropriate tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (PO4 and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), urea nitrogen (UN), and methemoglobin (MET-HGB). - Sacrifice and pathology:
- Non-fasted rodents submitted alive for necropsy were anesthetised by the inhalation of CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals.
GROSS PATHOLOGY: Yes
- Organs weights examined: The brain, liver, kidneys, heart, adrenals, testes, epididymides, thymus, and spleen.
HISTOPATHOLOGY: Yes
- Number of animals: A complete histopathology was performed on the control and high-dose group animals and all animals that died or were sacrificed in a moribund condition.
ORGANS AND TISSUES EXAMINED
Adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve - optic, duodenum, epididymides, esophagus, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/harderian glands, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina. - Statistics:
- Means and standard deviations (SD) were calculated for all continuous data. All parameters examined statistically (feed consumption is addressed below) were first tested for equality of variance using Bartlett's test (Winer, 1971). If the results from Bartlett's test were significant at alpha = 0.01, then the data for the parameter may be subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data was reviewed and an appropriate form of the data selected. The selected form of the data were then subjected to the appropriate parametric analysis as described under the title 'Statistical Analysis' in the field ‘any other information on materials and methods incl. tables’.
Feed consumption data was evaluated by Bartlett's test for equality of variances. Exploratory data analysis was performed by a parametric ANOVA and if significant at alpha = 0.05, was followed by Dunnett's test at alpha = 0.05, experiment-wise error.
Descriptive statistics only (means and SD’s) were reported for body weight gains, RBC indices, and WBC differential counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), and routinely excluded from feed consumption statistics. Other outliers may have been excluded only for documented scientifically sound reasons. DCO incidence scores were evaluated qualitatively.
If deemed necessary, more statistical tests were performed at the discretion of the study director. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Mortality:
- no mortality observed
- Description (incidence):
- No treatment related effects.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increases in platelet counts of males doses at ≥ 100 mg/kg bw/day. Effects considered to be non-adverse.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cholesterol values were increased in both males and females treated with ≥ 100 mg/kg bw/day. Effects considered to be non-adverse.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased absolute liver weight (≥ 100 mg/kg bw/day, males and females). Increased relative liver weights (males ≥ 100 mg/kg bw/day, females ≥ 500 mg/kg bw/day). Effects considered to be treatment related.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Hypertrophy + altered tinctorial properties of the hepatic lobule (500 or 1000 mg/kg bw/day males; 1 female 1000 mg/kg bw/day). Slight hepatocellular vacuolation in the periportal region consistent with fatty change (1000 mg/kg bw/day, males + females).
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: There was no mortality observed in the study animals.
- Cage-side and hand-held observations: One male and one female given 1000 mg/kg bw/day had decreased faeces on day 7. These observations resolved by days 8 and 9, for female and male, respectively, and were not attributed to treatment because of the transient nature of the observations.
- Detailed Clinical Observations: Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings. One male given 1000 mg/kg bw/day had an apparent mechanical injury to the tail prior to the start of the study. A separate male at this dose level also had decreased faeces on days 8 and 22. These observations were considered not to be treatment related due to the transient, infrequent occurrences.
BODY WEIGHT AND WEIGHT GAIN
There were no statistically-identified differences in the body weights of any treated groups when compared to their respective controls, nor were there any biologically significant differences in body weight gains.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: Variable numbers of male and female mice scratched feed during the study. This was not dose related, was attributed to normal animal activity and did not reflect a lack of diet palatability. There were no statistically-significant differences in the amount of feed consumed by any treated group when compared to their respective controls.
- Test Material Intake: The actual test material intake for males was 0, 10.8, 110, 538, and 1060 mg/kg bw/day; and for females was 0, 11.2, 113, 504, and 1140 mg/kg bw/day.
OPHTHALMOSCOPIC EXAMINATION
Examinations performed on all animals pre-exposure and at the termination of the study revealed that all animals were within normal limits.
HAEMATOLOGY
The platelet counts of both males and females treated at ≥ 100 mg/kg bw/day were greater than the controls and statistically identified (both sexes combined for analysis), see Table 1 for summary.
The mean platelet counts for males given ≥ 10 mg/kg bw/day and females given ≥ 100 mg/kg bw/day were outside of the historical control values. Platelet counts for males given ≥ 100 mg/kg bw/day were interpreted to be treatment-related because the means were clearly different from the control mean, the standard deviations were similar across all dose levels, and the values were outside of the historical control data. However, the differences between control and treated animals were interpreted not to be toxicologically significant, because there were no adverse effects associated with this difference in platelet counts. Platelet counts for females given ≥ 100 mg/kg bw/day were interpreted not to be treatment related because the statistical differences in females were most likely an artifact of the numerical differences in males. The difference between the high-dose and control means for the females was approximately half of the difference between the high-dose and control means for the males. This interpretation was further supported by an evaluation of the standard deviation that demonstrated a greater degree of variability in the control female group than any of the treated groups. A visual examination of the variation in individual animal platelet values indicated an approximate 70 % variation of platelet values within the control group.
Males given 500 or 1000 mg/kg bw/day and females given 1000 mg/kg bw/day had WBC counts that were lower than the controls but were not statistically identified. The apparent difference in WBC count of these mice were within the laboratory historical control range, while the concurrent control values for both males and females were outside the historical range. Therefore, the decreased total leukocyte counts were interpreted not to be treatment related. There were no additional alterations of significance.
CLINICAL CHEMISTRY
Cholesterol values in both males and females treated with ≥ 100 mg/kg bw/day were greater than the control values and statistically identified (both sexes combined for analysis), see Table 2 for summary.
The mean cholesterol values for each sex were outside the respective historical control values. These differences were attributed to treatment but were interpreted not to be an adverse effect. There were no additional alterations of significance.
ORGAN WEIGHTS
The absolute liver weights of animals treated with 100, 500 or 1000 mg/kg bw/day were greater than the controls and statistically identified, see Table 3 for summary.
The relative liver weights of males in the 100, 500, or 1000 mg/kg bw/day groups and females in the 500 or 1000 mg/kg bw/day groups were also greater than the controls and statistically identified. These absolute and relative liver weights were outside the historical control values (males: absolute range of 1.850-2.071, relative range of 5.413-6.019; females: absolute range of 1.329-1.588, relative range of 4.843-5.511, data in study file) and were attributed to treatment with the test material.
The absolute brain weights for males and females combined given 500 mg/kg bw/day and the relative brain weight for males given 100 mg/kg bw/day and females given 1000 mg/kg bw/day were lower than the controls and statistically identified. The differences in the 100 and 500 mg/kg bw/day groups were not dose related and were not attributed to treatment. The lower relative brain weight of females given 1000 mg/kg bw/day was attributed to the slightly greater body weight of these animals and was also interpreted not to be treatment related. There were no additional alterations of significance in organ weights.
GROSS PATHOLOGY
The only gross pathologic observations consisted of cloudy corneas in a few females from each dose group and a fractured tail in a male given 1000 mg/kg bw/day. These alterations were interpreted to spontaneous occurring or associated with accidentally injury and were not associated with exposure to the test material.
HISTOPATHOLOGY: NON-NEOPLASTIC
Males given 500 mg/kg bw/day and males and females given 1000 mg/kg bw/day had treatment-related effects in the liver, see Table 4 for summary.
Liver effects in the majority of males given 500 or 1000 mg/kg bw/day consisted of a hypertrophy and altered tinctorial properties (increased eosinophilia) of the hepatocytes in the centrilobular/midzonal or panlobular region of the hepatic lobule. In addition, two males given 500 or 1000 mg/kg bw/day had a very slight vacuolation of hepatocytes in the periportal region of the hepatic lobule that was consistent with fatty change. Three females given 1000 mg/kg bw/day also had a similar vacuolation of periportal hepatocytes. Centrilobular/midzonal hepatocytes of one male given 0 mg/kg bw/day, one male given 100 mg/kg bw/day, and one female given 1000 mg/kg bw/day also had hypertrophy with altered cytoplasmic staining. The effects in males were not attributed to treatment, whereas the effect in the high-dose female was possibly treatment related.
There were no treatment-related microscopic changes in any of the other tissues examined. All other observations were considered to be spontaneously occurring or accidentally induced, and not associated with exposure to the test material. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 10 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Based on the increases in platelet count and cholesterol level in males at ≥ 100 mg/kg bw/day, and increases in liver weights in both males and females given ≥ 100 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Based on the lack of histopathological findings accompanying the liver weight increases.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the no-observed-effect level (NOEL) and no-observed-adverse-effect level (NOAEL) were 10 and 100 mg/kg bw/day, respectively.
- Executive summary:
The subacute toxicity of the test material was assessed in a 28 week dietary toxicity study using CD-1 mice. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 407, EU Method B.7 and EPA OPPTS 870.3050.
Five male and five female mice per group were given test diets formulated to supply 0, 10, 100, 500, or 1000 mg/kg bw/day for 28 days. Cage-side clinical observations, detailed clinical observations, body weights, feed consumption, haematology, clinical chemistry, and organ weights were evaluated. In addition, a gross necropsy was conducted with extensive histopathologic examination of tissues.
The liver was the primary organ affected by the test material. Liver weights of males and females given ≥ 100 mg/kg bw/day were increased and statistically identified. Treatment-related microscopic changes occurred in the livers of males given 500 or 1000 mg/kg bw/day and one female given 1000 mg/kg bw/day and consisted of centrilobular/midzonal to panlobular hepatocellular hypertrophy with altered tinctorial properties. A few of the males given 500 or 1000 mg/kg bw/day and females given 1000 mg/kg bw/day also had slight hepatocellular vacuolation in the periportal region consistent with fatty change.
Platelet counts of males given ≥ 100 mg/kg bw/day and cholesterol levels of males and females given ≥ 100 mg/kg bw/day were increased and statistically identified. These differences were attributed to treatment but were not adverse effects. There were no treatment-related effects in body weights, body weight gains, feed consumption, ophthalmologic, clinical, and gross pathological observations.
Under the conditions of the test, the no-observed-effect level (NOEL) and no-observed-adverse-effect level (NOAEL) were 10 and 100 mg/kg bw/day, respectively.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 12 July 2001 - 25 March 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF Testing Guidelines Subchronic Oral Toxicity Study
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: Housed 2 per cage during acclimatisation and individually during the main study in stainless steel cages.
- Diet: Certified rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Animals were acclimated to the laboratory for one week prior to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):40 - 70 %
- Air changes (per hr): Approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: 17 July 2001 To: 17 October 2001 (males) and 18 October 2001 (females). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: Premixes were mixed periodically throughout the study based on stability data. Diets were prepared weekly based upon the most recent body weight and feed consumption data.
- Mixing appropriate amounts with: Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low-dose female and high-dose male test material-feed mixtures were determined pre-exposure, near the middle and end of the study. Aliquots were taken from multiple areas within the containers. The method for analysis of the test material in feed was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Stability: A previous 13-week dietary toxicity study in rats has shown the test material to be stable for at least 42 days in the feed at concentrations of 0.005 % up to a 5 % premix. The concentrations of test material in the diets fed in this 90-day study were within this range.
- Concentration Verification: Analyses of all dose levels, plus control and premix were initiated pre-exposure, near the middle and end of the study. The method used for analysing the test material in feed was a solvent extraction method followed by quantitation using high-performance liquid chromatography (HPLC) with mass spectrometric detection and internal as well as external standards.
- Retainer Samples: Reference samples (one/sex/dose/mix and premix) were retained and stored at ambient temperature in sealed vials in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- 90 consecutive days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 3 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - No. of animals per sex per dose:
- Ten per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Data from a 28-day study indicated that mice should tolerate a high-dose of 1000 mg/kg bw/day based on the absence of adverse clinical observations and the lack of an effect on feed consumption or body weights. The high-dose was expected to produce liver effects in both sexes. The other dose levels (30 and 300 mg/kg bw/day) were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low-dose of 3 mg/kg/day was expected to be a NOEL.
- Dosing route rational: Probable routes of human exposure to the test material would be via accidental ingestion during application or manufacture. Thus, administration of the test material to rats via the diet represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations included where possible: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were conducted at pre-exposure and weekly throughout the study. The detailed clinical observation (DCO) was conducted on all animals, at approximately the same time each examination day.
- DCO included: Cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: All mice were weighed during the pre-exposure and weekly during the remainder of the study. Body weight gains were also calculated.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected pre-exposure and at least weekly thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) /(No. of days in measurement cycle)
- Test material intake (TMI): TMI was calculated using actual feed concentrations, body weights and feed consumption data in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All eyes were examined prior to the scheduled necropsy using indirect ophthalmoscopy.
- Dose groups that were examined: All animals were examined.
- Method: One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus at the scheduled necropsy.
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: No
- How many animals: All animals
- Sample preparation: Blood samples for a complete blood count were mixed with ethylenediamine- tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and archived. Haematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
- Parameters checked: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, RBC indices (MCH, MCV and MCHC) and methemoglobin concentration.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus at the scheduled necropsy.
- Animals fasted: No
- How many animals: All animals
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN). - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- Organ weights: brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus and spleen.
HISTOPATHOLOGY: Yes
- Tissues examined: Adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, esophagus, eyes, gallbladder, gross lesions, heart, ileum, jejunum, kidneys,lacrimal/harderain glands, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina. - Statistics:
- Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, clinical chemistry data, and appropriate haematologic data were evaluated by Bartlett's test (α = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at α = 0.05, the ANOVA were followed respectively by Dunnett's test (α= 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (α = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. DCO incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (α= 0.05; Bruning and Kintz, 1987). Data collected at different time points were analysed separately.
Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (α= 0.02; Grubbs, 1969), but routinely excluded only from feed consumption calculations. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) were greater than the nominal alpha levels. Therefore, the final interpretation of the data will consider statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Mortality occured (males given ≥ 30 mg/kg bw/day, females given ≥ 300 mg/kg bw/day). No treatment related effects in male and female cage side observations or detailed clinical observations.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Mortality occured (males given ≥ 30 mg/kg bw/day, females given ≥ 300 mg/kg bw/day). No treatment related effects in male and female cage side observations or detailed clinical observations.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Alterations in body weights and body weight gains were observed, unclear if these effects were treatment related or not.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased cholesterol (females given 1000 mg/kg bw/day); increased enzyme activities ALT and AST and very slight microscopic hepatocelluar necrosis (females given 1000 mg/kg bw/day).
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increases in absolute liver weights (males given ≥ 300 mg/kg bw/day and females given ≥ 30 mg/kg bw/day); increased relative liver weights (males given ≥ 3 mg/kg bw/day and females given ≥ 30 mg/kg bw/day).
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Pale foci in the liver corresponding to very slight foci of hepatic necrosis (single incidence in a female given 1000 mg/kg bw/day).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment realted effects observed (males given ≥ 3 mg/kg bw/day and females given ≥ 30 mg/kg bw/day) see 'Details on results' below.
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: No mortality occurred in the control groups. One male given 30 mg/kg bw/day, one male given 300 mg/kg bw/day and four males given 1000 mg/kg bw/day died or were euthanatised prior to the scheduled necropsy. Similarly, three females given 300 mg/kg bw/day and five females given 1000 mg/kg bw/day, died prior to the scheduled necropsy. The first deaths occurred on day 36 in a female given 1000 mg/kg bw/day and a male given 30 mg/kg bw/day. The remainder of the deaths occurred between days 40 and 90.
- Cage-side observations: Examinations performed on all animals revealed no abnormal observations.
- DOC: Examinations performed on all animals pre-exposure and at termination of the in-life phase of the study revealed that a few males and females given ≥ 300 mg/kg bw/day had isolated convulsions from day 77 until the end of the study. Convulsions occur fairly commonly in this strain of mouse when they are handled, however, convulsions were not observed in any of the control male or female mice nor were they observed in any of the mice which died or were moribund. Convulsions were interpreted not to be treatment related. In addition, mice which died or were moribund did not have any significant clinical observations. All other observations were also interpreted not to be treatment related.
BODY WEIGHT AND WEIGHT GAIN
There were no statistically identified differences in the body weights of any treated male group when compared to the control. However, males given 30, 300 or 1000 mg/kg bw/day gained approximately 4, 18 and 15 % less weight, respectively, than the controls by the end of the study. In contrast to the decrease body weight gain in males, females given 3, 30, 300 or 1000 mg/kg bw/day weighed more than the controls and had body weight gains of approximately 23, 33, 60 and 60 % greater than the control, respectively. These differences in body weight of females given 300 or 1000 mg/kg bw/day were occasionally statistically identified. It was not clear if the alterations in body weights and body weight gains were treatment related.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
Test Material Intake and Parts per Million in Feed: Calculated test material intake for males and females indicated that they received the targeted concentrations of test material at all dose levels.
OPHTHALMOSCOPIC EXAMINATION
Examinations performed on all animals prior to the study and at termination revealed isolated instances of pale fundus or cloudy cornea that were not dose related and were interpreted not to be treatment related.
HAEMATOLOGY
There were no treatment related changes in any of the haematologic parameters for males or females at any dose level.
The methaemoglobin level for males given 1000 mg/kg bw/day was slightly higher than the controls, however, there were no indications of an anemia or Heinz body formation in individual male mice which had the highest methaemoglobin levels that suggested that the difference in methaemolgobin concentration was significant. Therefore, the methaemoglobin level in males given 1000 mg/kg bw/day was interpreted not to be affected by treatment with the test material.
CLINICAL CHEMISTRY
The mean cholesterol value for females given 1000 mg/kg bw/day was slightly higher than the control value and statistically identified. This difference was interpreted to be treatment related because the value was outside of the historical control values and was statistically identified, however, the value of 139 represented the mean of only five mice and only one of the values for these mice was clearly high (182 mg/dL).
The ALT and AST activities for females given 1000 mg/kg bw/day were also higher than the concurrent and historical control values but were not statistically identified.
These higher enzyme activities were due to increases in two females (5521 and 5526), which also had very slight microscopic hepatocelluar necrosis and were interpreted to be treatment related.
There were no additional statistically identified significant changes in any of the clinical chemistry parameters for males or females when compared to their respective controls, with the exception of a higher cholesterol in males given 30 mg/kg bw/day. This higher value was not attributed to the test material because a similar effect was not evident at the higher dose levels.
See Table 2.
ORGAN WEIGHTS
Treatment-related increases in absolute and relative liver weights occurred in males given ≥ 300 mg/kg bw/day and females given ≥ 30 mg/kg bw/day. The relative liver weights of males given 3 or 30 mg/kg bw/day were also increased compared to control values and statistically identified.
The absolute kidney weights of females given 300 or 1000 mg/kg bw/day were higher than the controls and statistically identified. These differences were not attributed to treatment with the test material because the body weights of these mice were higher than the control value and the relative kidney weights were not significantly different from the controls.
The absolute spleen weight of females given 1000 mg/kg bw/day was higher than the control value and statistically identified. This weight difference was not attributed to the test material because the relative weight of the spleen was not different from the control value and there were no lesions microscopically identified in the spleen of these mice.
See Table 3.
GROSS PATHOLOGY
The only treatment-related gross observation was the single incidence of pale foci in the liver of one female (5526) given 1000 mg/kg bw/day. These pale foci corresponded to very slight foci of hepatic necrosis which were attributed to treatment. There were no other treatment-related gross pathologic observations in mice that died or were moribund, or in mice that survived until the scheduled termination of the study. All other gross pathologic observations were considered to be spontaneous alterations or nonspecific and were not associated with exposure to the test material.
HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic effects were restricted to the liver and affected males given ≥ 3 mg/kg bw/day and females given ≥ 30 mg/kg bw/day.
Centrilobular to midzonal hypertrophy (increased size) and altered tinctorial properties (increased eosinophilia) of the hepatocyte cytoplasm occurred in a few males given 3 or 30 mg/kg bw/day and the majority of males and females given 300 or 100 mg/kg bw/day.
The majority of males and a few females given 300 or 1000 mg/kg bw/day had very slight or slight vacuolation of the periportal hepatocytes, consistent with fat accumulation.
Foci of necrotic hepatocytes of a very slight degree were infrequently observed in the livers of a few males given 300 or 1000 mg/kg bw/day and four females given 1000 mg/kg bw/day. Inflammatory cells were noted adjacent to the necrotic hepatocytes. A very slight inflammation was also present in the livers of males given 300 or 1000 mg/kg bw/day, in the absence of necrotic hepatocytes. This inflammation was characterised by the accumulation of macrophages and variable numbers of neutrophils.
See Table 4. - Dose descriptor:
- LOEL
- Effect level:
- 3 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Based on increased liver weights.
- Dose descriptor:
- LOEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Based on increased liver weights.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 3 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Based on increased liver weights observed at 30 mg/kg bw/day.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the lowest-observed-effect level (LOEL) for males and females was 3 and 30 mg/kg bw/day, respectively, based on increased liver weights. The no-observed-effect level (NOEL) for females was 3 mg/kg bw/day and was not determined for males.
- Executive summary:
The subchronic toxicity of the test material was assessed in a 90 week dietary toxicity study using CD-1 mice. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 408, EU Method B.26, EPA OPPTS 870.3100 and JMAFF testing guidelines.
Ten male and ten female mice per dose level were given diets formulated to supply 0, 3, 30, 300 or 1000 mg test material/kg bw/day for 90 days. Parameters evaluated included daily observations, detailed clinical observations, ophthalmic examinations, body weight, feed consumption, clinical pathology, organ weights, and gross and histopathologic examinations.
A treatment-related increase in mortality occurred in males given ≥ 30 mg/kg bw/day and females ≥ 300 mg/kg bw/day, with the total number of dead animals in the 30, 300 and 1000 mg/kg bw/day groups being 1/20, 4/20 and 9/20, respectively. Males given 3, 30, 300 or 1000 mg/kg bw/day had increases in relative liver weight of approximately 7, 10, 40 and 39 %, while females given 3, 30, 300 or 1000 mg/kg bw/day had increases in relative liver weight of approximately 5, 14, 42 and 44 %, respectively. Treatment-related microscopic effects occurred in the livers of males given ≥ 3 mg/kg bw/day and females given ≥ 300 mg/kg bw/day. Centrilobular to midzonal hypertrophy and altered tinctorial properties of the hepatocyte cytoplasm occurred in a few males given 3 or 30 mg/kg bw/day and the majority of males and females given 300 or 1000 mg/kg bw/day. The increases in liver weight and hypertrophy were likely secondary effects related to the induction of hepatic enzymes required for the metabolism of the test material. Hepatocytes in the periportal region of the hepatic lobule also had the accumulation of vacuoles which appeared to be lipid, and chronic inflammation occurred with and without the presence of necrotic hepatocytes.
The vacuolation and inflammation/necrosis were noted in males given ≥ 300 mg/kg bw/day and females given 1000 mg/kg bw/day and were interpreted to be degenerative effects attributed to the test material. The lowest-observed-effect level (LOEL) for males and females was 3 and 30 mg/kg bw/day, respectively, based on increased liver weights. The no-observed-effect level (NOEL) for females was 3 mg/kg bw/day and was not determined for males.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 January 2001 - 03 January 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF Testing Guidelines Subchronic Oral Toxicity Study
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 weeks.
- Housing: Individually, in stainless steel cages.
- Diet: Rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Acclimated to the laboratory for one week prior to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 - 22.7 °C
- Humidity (%): 45 - 52 %
- Air changes (per hr): Approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m.
IN-LIFE DATES: From: 25 January 2001 To: 26 April 2001 (males) and 27 April 2001 (females), respectively (test days 92 and 93). Recovery group animals were necropsied on 25 May 2001 (test day 121). - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Premixes were mixed periodically throughout the study based on stability data. Diets were prepared weekly based upon the most recent body weight and feed consumption data.
- Mixing appropriate amounts with (Type of food): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low-dose female and high-dose male test material-feed mixtures were determined pre-exposure, near the middle, and end of the study. Aliquots were taken from multiple areas within the containers. The method for analysis of the test material in feed was a solvent extraction method followed by analysis using liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Stability: The stability of the test material in rodent feed was determined pre-exposure over the concentration range used in this study (low-dose female diet and the high-dose male diet).
- Concentration verification: Analyses of all dose levels for the test material were conducted pre-exposure, near the middle, and end of the study. The method used for analysing the test material in feed was as described above.
- Retainer samples: Reference samples (one/sex/dose/mix and premix) were retained and stored at ambient temperature in sealed vials in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- At least 90 consecutive days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - No. of animals per sex per dose:
- Ten per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose of 1000 mg/kg bw/day was selected based on results of the 28-day study. The high-dose was expected to produce effects in the liver and kidney. The mid- and low-dose levels of 500, 100, and 10 mg/kg bw/day were expected to provide dose-response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).
- Dosing route rational: Probable routes of human exposure to the test material would be via accidental ingestion during application or manufacture. Thus, administration of the test material to rats via the diet represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program.
- Post-exposure recovery period in satellite groups: Additional groups (ten/sex/dose) were given exposed to the test material at either 0 or 1000 mg/kg bw/day for 90 days and were then given control feed for an additional 28 days to assess the reversibility of treatment-related effects induced following exposure to the test material. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day.
- Cage side observations (where possible): Skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted pre-exposure and weekly throughout the study. DCO were conducted on all animals, at approximately the same time each examination day. The data for all in-life parameters were separated between the rats designated for the main and the recovery groups to facilitate computerized data management.
- DCO examinations: Cage-side, hand-held and open-field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and at least weekly during the remainder of the study. Body weight gains were also calculated.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected pre-exposure and at least weekly thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder - final weight of feeder) /[(No. of days in measurement cycle)(No. of animals per cage)]
- Test material intake: TMI was calculated using actual feed concentrations, body weights and feed consumption data in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: A veterinarian examined the eyes pre-exposure and prior to the scheduled necropsy (main group only) using indirect ophthalmoscopy.
- Dose groups that were examined: The eyes of all animals were examined.
- Method: One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during necropsy through a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus following exposure to CO2, at the scheduled necropsy.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation:
> Complete blood count: Blood samples for a complete blood count (main group only) were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright’s stain, and archived. Haematologic parameters were assayed using a Technicon H•1E Haematology Analyser.
> Coagulation: Blood samples for coagulation were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an MLA Electra 700, Automatic Coagulation Timer. Prothrombin time was also evaluated in male rats from the recovery group.
- Parameters checked:
> Complete blood count: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, RBC indices (MCH, MCV and MCHC), and methaemoglobin concentration.
> Coagulation: Prothrombin time (PT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus following exposure to CO2, at the scheduled necropsy.
- Animals fasted: Yes
- How many animals: All animals.
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN).
> Recovery group: Alkaline phosphatase, aspartate aminotransferase, glucose, total protein and cholesterol were evaluated in males from the recovery groups and alkaline phosphatase, glucose, albumin, cholesterol, and calcium were evaluated in females from the recovery groups.
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the week prior to necropsy (~16-hour period). Urine was also collected by manual compression of the bladder prior to the necropsy for characterization of the microsediment.
A timed urine volume collection with complete urinalysis was conducted as previously described, but without microscopic observations of the microsediment, from the recovery groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Colour, appearance and specific gravity (refractometer), and urine volume. Semiquantitative analysis of: pH, bilirubin, glucose, proteins, ketones, blood, and urobilinogen. - Sacrifice and pathology:
- Fasted rodents were exposed to CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re- examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle.
GROSS PATHOLOGY: Yes
- Dose groups examined: All animals.
- Organ weights examined: The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated. The brain, liver, and kidneys from recovery group males, and the liver, adrenals, and spleen from recovery groups females were also weighed.
HISTOPATHOLOGY: Yes
- Dose groups examined: All control and high-dose group animals. The livers from the recovery groups were also histologically examined.
- Tissues examined: Adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina. - Other examinations:
- ENZYMOLOGY
- Sample collection: Representative samples of liver were collected during the necropsy after approximately 90 days of exposure to the test material.
- Sample collection: Tissues were flash frozen in liquid nitrogen, and stored at -80 °C. Frozen tissues were subsequently processed by appropriate methods to obtain microsomal fractions from each animal. The relative activities of several mixed function oxidase enzymes (MFO - CYP1A1, CYP1A2, CYP2B1/2, and CYP2E1) were measured.
> Microsome Isolation
- Dose groups examined: Five animals per sex from the control and high dose groups were examined.
- Method: Total microsomal proteins for individual animal and pooled samples were determined using the Pierce BCA™ method.
> In Vitro Enzyme Assays
- Dose groups examined: Animals in the high dose group only.
- Method: The activities of selected MFO enzymes were measured in vitro using generally accepted methodologies. CYP1A1, CYP1A2, and CYP2B1/2 activities were measured as ethoxyresorufin (EROD), methoxyresorufin (MROD) and pentoxyresorufin (PROD) O-dealkylase activities, respectively, using the microplate fluorometric method outlined by Kennedy and Jones (1994). These methods involved the measurement of the in vitro O-dealkylation of 7-alkoxyresorufins to a highly fluorescent product, resorufin, by relatively closely related microsomal MFOs (reviewed by Burke and Mayer, 1983). CYP2E1 activity was measured as the hydroxylation of ρ-nitrophenol (p-NPH) using the spectrophotometric method outlined by Reinke and Moyer (1985).
ELECTRON MICROSCOPY
- Dose groups examined: Three rats/sex from the control and high-dose groups.
- Sample collection/preparation: Liver samples (left lateral lobe) were diced into approximately 1 mm³ cubes to aid rapid fixation and placed in phosphate buffered solution of 2 % glutaraldehyde/2 % formaldehyde for 1-2 days on a rotator in a cold room. Tissue specimens were post fixed in 1 % osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in epon/araldite epoxy resin. Thick sections (1-2 µ) were cut with a diamond microtome, and stained with toluidine blue. The centrilobular areas of sections demonstrating well defined hepatic lobules were selected using light microscopy, and thin sections (60-90 mµ) were cut from the corresponding blocks using a Sorvall®MT-2B ultramicrotome.
These sections were picked up on 200 mesh copper grids, air dried, and stained with uranyl acetate and lead citrate. Tissue specimens from 2-3 rats/sex/dose were evaluated using a Hitachi H-600 transmission electron microscope and representative hepatocytes were digitally photographed.
- Evaluation: Electron micrographs were printed and evaluated by a veterinary pathologist.
URINE METABOLITES
- Dose groups examined: Pooled sample from each dose group/sex.
- Sample collection: Urine was collected, by manual compression of the bladder prior to the necropsy, for preliminary metabolite identification.
- Analysis: Urine samples were analysed using high performance liquid chromatography/negative electrospray ionization/mass spectrometry. - Statistics:
- Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation and appropriate haematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA were followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise α level was reported for these two tests. DCO incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analysed separately. Descriptive statistics only (means and standard deviations) were reported for body weight gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed consumption calculations. Outliers may have been excluded from other analyses only for documented, scientifically sound reasons.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) were greater than the nominal alpha levels. Therefore, the final interpretation of the data will consider statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No toxicologically significant effects.
- Mortality:
- no mortality observed
- Description (incidence):
- No toxicologically significant effects.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduced body weight and body weight gains (males and females given ≥ 100 mg/kg bw/day).
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No toxicologically significant effects.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No toxicologically significant effects.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased prohrombin time (males given ≥ 100 mg/kg bw/day).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased ALP and cholesterol (males and females given ≥ 100 mg/kg bw/day); deceased AST (males and females ≥ 100 mg/kg bw/day); decreased glucose (males ≥ 100 mg/kg bw/day and females at 1000 mg/kg bw/day).
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- No toxicologically significant effects.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased liver weights (males and females given ≥ 100 mg/kg bw/day); increased relative liver weights (males and females given ≥ 10 mg/kg bw/day); increased adrenal weights and relative adrenal weights (females given ≥ 100 mg/kg bw/day).
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No toxicologically significant effects.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Microscopic liver hypertrophy with increased eosinophilia, centrilobular to midzonal (males given ≥ 10 mg/kg bw/day and females given ≥ 100 mg/kg bw/day).
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- > MAIN GROUP
CLINICAL SIGNS AND MORTALITY
- Mortality: There was no mortality.
- Cage-Side Observations: On day 82, one female given 10 mg/kg bw/day had maloccluded incisors and blood in the cage. These findings were interpreted not to be treatment related because of the low incidence and lack of a dose-response relationship.
- Detailed Clinical Observations: Examinations performed on all animals revealed a low and sporadic incidence of palpebral closure, cloudy eye, maloccluded teeth, and perineal and periocular soiling. Increased lacrimation was also observed but occurred more in controls than in rats exposed to the test material. Two females given 500 mg/kg bw/day had ulcerated areas (palpable masses) on the muzzles that were transient. All of these observations were low in incidence, not statistically identified, or dose related and were interpreted not to be related treatment.
BODY WEIGHT AND WEIGHT GAIN
Male and female rats given 100, 500, or 1000 mg/kg bw/day weighed less than the controls from approximately days 43 (males) and 43 (females) until the end of the study. The differences in body weights were only 3 - 4 % lower than the controls at the end of the study, were occasionally statistically identified, and were not dose related. Differences in body weights were also reflected in lower body weight gains for rats given 100, 500, or 1000 mg/kg bw/day. The differences in body weight gains were 4.7 - 8.8 % (males) and 6.9 - 13.4 % (females) lower than the controls at the end of the study. These differences in body weights and body weight gains, although not dose related were attributed to the test material.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: Male rats given 100, 500, or 1000 mg/kg bw/day had a slightly lower feed consumption compared to the controls during the later half of the dosing phase of the study, and these differences were occasionally statistically identified. Differences in feed consumption were consistent with the slightly lower body weights of these male rats. Slightly lower feed consumption values were also noted for females given 10, 100, 500, or 1000 mg/kg bw/day, starting the first month of the study and occasionally occurring during the remainder of the dosing phase of the study. These differences were occasionally statistically identified, however, they were not dose related and were interpreted not to be a clear effect of treatment.
- Test Material Intake: Male and female rats given 10, 100, 500, or 1000 mg/kg bw/day received levels of test material that were acceptably close to the targeted concentrations for each dose group.
OPHTHALMOSCOPIC EXAMINATION
The eyes of all rats were within normal limits at the pre-exposure examination. Variable numbers of males and two females had a cloudy cornea at the end of the study, with the controls and the lower-dose levels having the highest incidences. A pale fundus and periocular soiling were infrequently observed in isolated rats from the main group at the end of the study. These observations were interpreted not to be treatment related due to the low incidence and lack of a dose-response relationship.
HAEMATOLOGY
- Haematology: Females given 10, 100, 500 or 1000 mg/kg bw/day had hemoglobin concentrations that were slightly lower than the control value and statistically identified. These differences were interpreted not to be treatment related because the values for rats given the test material were well within the historical control range and the concurrent controls were slightly higher than the historical control range. There were no significant changes in any of the haematologic parameters for male rats or any other changes for female rats.
- Prothrombin Time: Male rats given 100, 500, or 1000 mg/kg bw/day had prothrombin times that were greater than the controls, and the values for the 500 and 1000 mg/kg bw/day groups were statistically identified.
Prothrombin time differences at 100, 500, and 1000 mg/kg bw/day were interpreted to be treatment related. However these effects were not toxicologically significant, because there was no indication that these prolonged prothrombin times were associated with any clinical bleeding abnormalities. There were no significant differences in the prothrombin times of females at any dose level.
CLINICAL CHEMISTRY
Male and female rats given 100, 500, or 1000 mg/kg bw/day had higher alkaline phosphatase (ALP) activities and cholesterol values, and lower aspartate aminotransferase (AST) activities than the controls. Glucose levels of males given 100, 500, or 1000 mg/kg bw/day and females given 1000 mg/kg bw/day were also slightly lower than the controls. These differences were statistically identified, except for the female and the 500 mg/kg bw/day male AST activities, interpreted to be treatment related, but not adverse based on the absence of clinical disease, and were likely secondary to alterations in liver metabolism.
A number of other clinical chemistry parameters were statistically different from the controls but were interpreted not to be treatment related because the values were:
1) Within or consistent with the historical control range (higher total protein (TP) for males given 500 or 1000 mg/kg bw/day; lower total bilirubin for females given ≥10 mg/kg bw/day),
2) Lacked a dose-response relationship (lower potassium for females given 500 mg/kg bw/day; lower albumin and calcium for females given ≥100 mg/kg bw/day),
3) Concurrent control value was not consistent with historical controls (total bilirubin for females),
4) The differences between the control and treated groups were minor (lower albumin and calcium for females given ≥ 100 mg/kg bw/day).
URINALYSIS
Urine volumes of male rats given 10, 100, 500, or 1000 mg/kg bw/day and female rats given 100, 500, or 1000 mg/kg bw/day were higher than the controls and some of these differences were statistically identified. Values for females were somewhat dose related, but the values were not dose related for males. A historical control urine volume for males indicated that the concurrent control value was low and the differences were not meaningful. Urine volume for females given 100, 500, or 1000 mg/kg bw/day were higher than the historical control value and could possibly be treatment related. Lower urine specific gravities were noted for males given 10, 100, 500, or 1000 mg/kg bw/day and females given 100, 500 or 1000 mg/kg bw/day and were statistically identified. Specific gravity values for the test material males and females were within or in close proximity to the historical control values and indicated that the differences in specific gravity were not biologically significant. In addition, microscopic treatment-related changes were not observed in the kidneys of either male or females rats given the test material. Urine pH appeared more alkaline in males and females given the test material. These differences may be associated with the excretion of alkaline metabolites of the test material, presence of compounds that interfere with the pH test strip, or reflect the normal variability in this parameter.
ORGAN WEIGHTS
The final body weights of male and female rats given 100, 500, or 1000 mg/kg bw/day were slightly lower than the controls (2 – 5 %) and were attributed to treatment, however; only the body weights of males given 500 mg/kg bw/day were statistically identified. Liver weights (absolute and relative) of males and females given 100, 500, or 1000 mg/kg bw/day were increased compared to the controls and statistically identified. The relative liver weights of males and females given 10 mg/kg bw/day were also increased and statistically identified. The adrenal gland weights (absolute and relative) of females given 100, 500, or 1000 mg/kg bw/day were increased compared to the controls and statistically identified. These organ weight changes were interpreted to be treatment related.
The relative liver weights of males given 10, 100, 500, or 1000 mg/kg bw/day were 7.4, 20.7, 21.2, or 20.5 % larger than the controls; whereas, the relative liver weights of females given 10, 100, 500, or 1000 mg/kg bw/day were 7.8, 34.4, 43.3, or 40.3 % larger than the controls. The percentage increases in liver weights were not proportional to the dose of test material administered and clearly indicated a lack of a dose relationship in the increased liver weights of males and females given 100, 500, or 1000 mg/kg bw/day. A similar lack in the proportionality was also evident in the increases in adrenal gland weights (absolute and relative) in female rats given 100, 500, or 1000 mg/kg bw/day.
The following organ weights were higher or lower than the controls in rats given the test material and were frequently statistically identified:
1) Lower absolute epididymal weights in males given 500 or 1000 mg/kg bw/day,
2) Higher relative heart and spleen weights of females given 100, 500, or 1000 mg/kg bw/day,
3) Higher relative thymus weight of females given 1000 mg/kg bw/day,
4) Higher relative kidneys weight of males given 100, 500, or 1000 mg/kg bw/day and females given 500 mg/kg bw/day,
5) Higher relative brain weights of males and females given 100, 500, or 1000 mg/kg bw/day.
These differences were interpreted to be secondary to the lower body weights of these rats and were interpreted not to be a direct effect of treatment with the test material.
GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were interpreted to be spontaneous alterations (cloudy corneas, strangulated fat, liver hernia, gingival abscess, malocclusion, and facial soiling).
HISTOPATHOLOGY: NON-NEOPLASTIC
The liver was identified as a primary target organ in males given 10, 100, 500 or 1000 mg/kg bw/day and female rats given 100, 500, or 1000 mg/kg bw/day. Microscopic changes were characterized by an increase in hepatocellular size (hypertrophy) in the centrilobular and midzonal region of the hepatic lobule. In addition, these hepatocytes had an increase in eosinophilic staining of the cytoplasm (altered tinctorial properties).
All other observations were interpreted to be the result of spontaneous alterations, unassociated with the test material.
ELECTRON MICROSCOPY
Male and female rats given 1000 mg/kg bw/day had slightly increased amounts of smooth endoplasmic reticulum. All other organelles appeared qualitatively and quantitatively normal.
LIVER ENZYMES
The activities of the hepatic CYP1A subfamily of mixed function oxygenase enzymes were statistically identified as elevated in high-dose group rats relative to controls. A moderate 4.4 - 4.5 fold increase in MROD, indicative of CYP1A2 activity, and a 2.0 - 2.2 fold increase in EROD, indicative of CYP1A1 activity, was observed in high-dose males and females. In addition, minimal, yet occasionally statistically identified increases (1.2 - 1.7 fold) in PROD and p-NPH activities, indicative of CYP2B1/2 and CYP2E1 activities, respectively, were also noted in high-dose group rats. Taken together, these changes in MFO activities correlated with increased liver weights, light and electron microscopic findings of treated rats and were consistent with an adaptive response to the increased metabolic load placed upon this organ.
CHEMICAL ANALYSIS OF URINE
Urine samples from the 500 and 1000 mg/kg bw/day males had test material levels above the low standard (0.091 µg/mL), which was set at the detection limit for this analysis. The amount of test material in these samples was less than 0.03 % of the administered dose. Samples were also analysed for the free aniline metabolite of the test material, however; no useful data were obtained from these analyses.
Urine samples from male and female rats given 1000 mg/kg bw/day were screened for potential metabolites based on the known metabolic pathway for a similar molecule. No definitive metabolite identification data were obtained from these samples due to the occurrence of numerous peaks in the chromatogram, which could not be discriminated. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 10 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the lowest-observed-effect level (LOEL) and no-observed-adverse effect level (NOAEL) were 10 mg/kg/day based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males. A no- observed-effect level was not determined.
- Executive summary:
The subchronic toxicity of the test material was assessed in a 13 week dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 408, EU Method B.26, EPA OPPTS 870.3100 and JMAFF testing guidelines subchronic oral toxicity study.
Ten male and ten female Fischer 344 rats per group were given test diets formulated to supply 0, 10, 100, 500, or 1000 mg/kg bw/day for at least 90 days. Additional groups (ten/sex) were given either 0 or 1000 mg/kg bw/day for 90 days and were then given control feed for an additional 28 days to assess the reversibility of treatment-related effects induced following exposure to the test material. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations, and hepatic mixed function oxidase levels.
Male and female rats given 100, 500, or 1000 mg/kg bw/day had decreases in body weights (3-6 %) and body weight gains (4.7-13.4 %) compared to the controls which lacked a dose-response relationship. Liver weights of male and female rats given 10, 100, 500, or 1000 mg/kg bw/day were increased and statistically identified. Liver weight increases in rats given 100, 500, or 1000 mg/kg bw/day were not proportional to the dose administered. Microscopic effects consisting of an increase in hepatocytes size with an increase in eosinophilic staining of the hepatocyte cytoplasm occurred in males given ≥ 10 mg/kgbw/day and females given ≥ 100 mg/kg bw/day. Enzyme determinations indicated that the increases in liver weights were associated with a modest induction of a number of liver enzymes. In addition, the electron microscopic examination of the livers indicated that the smooth endoplasmic reticulum of centrilobular hepatocytes was increased in amount. Other effects of no or minor toxicologic significance occurred in male and/or female rats given ≥ 100 mg/kg bw/day during the dosing phase of the study and consisted of: 1) increased adrenal gland weights (females only), 2) minor increases in alkaline phosphatase activity, 3) minor decreases in aspartate aminotransferase activity and glucose levels (primarily males), and minor increases in cholesterol and prothrombin time (males only).
Complete recovery of treatment-related effects during the dosing phase of the study occurred with the following parameters: microscopic hepatocellular hypertrophy (females), and numerous alterations in clinical pathology parameters. A few of the parameters affected during the dosing phase of the study partially reversed during the recovery phase of the study and included: liver weights and adrenal weight increases, urine pH (females), and microscopic hepatocellular hypertrophy (males). The lower body weights identified during the dosing phase of the study persisted throughout the recovery phase of the study indicating that this effect was not reversible in 28 days.
Under the conditions of the test, the lowest-observed-effect level (LOEL) and no-observed-adverse effect level (NOAEL) were 10 mg/kg/day based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males. A no- observed-effect level was not determined.
- Endpoint:
- chronic toxicity: oral
- Remarks:
- combined repeated dose and carcinogenicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 9 April 2002 - 22 April 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity) and EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies) and OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method Part B. (Combined Chronic Toxicity / Carcinogenicity Test) and EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF, Combined Chronic Toxicity/Oncogenicity Study and Subchronic Oral Toxicity Study) 1985
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 weeks.
- Housing: The animals were housed 2-3 per cage during acclimatisation. During the study animals were housed 2 per cage in in stainless steel cages.
- Diet: Certified rodent diet in meal form, provided ad libitum.
- Water: Municipal water, provided ad libitum.
- Acclimation period: Animals were acclimated to the laboratory for 7 days prior to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 to 22.8 °C, with the exception of one day when the temperature was 27.7 °C.
- Humidity (%): 40 to 69 %, with the exception of three occasions when the humidity was 38 %.
- Air changes (per hr): A 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m.
- Photoperiod (hrs dark / hrs light): approximately 12-15 times/hour.
IN-LIFE DATES: From: April 18, 2002 (males) and April 19, 2002 (females). To: Subchronic toxicity animals were necropsied July 18, 2002 (days 92 and 91 for males and females, respectively). The chronic toxicity animals were necropsied April 16 and 17, 2003 (day 364) for males and females, respectively. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
Diets were prepared by serially diluting a concentrated test material- feed mixture (pre- mix) with ground feed. Premixes were mixed periodically throughout the study based on stability data. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data. Subsequently, the concentrations of the test material in the feed were adjusted weekly for the first 13-weeks of the study and at 4-week intervals thereafter based upon the most recent body weight and feed consumption data. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Homogeneity: The homogeneity of the low-dose female and high-dose male test material- feed mixtures were determined prior to the start of dosing and at approximately 1.5, 3, 8, 12, 18, and 24 months. Additional analytical analyses were conducted at approximately 8, 9, 14, and 20 months to verify homogeneity of the diet mixes.
- Stability: Previous 90-day toxicity study in rats (Yano and Dryzga, 2002) demonstrated that the test material was stable for at least 42 days in rodent chow at concentrations ranging from 0.005 % to 5 %. In this study, an additional stability evaluation was conducted at 0.0001 % to cover the concentration ranges of the administered diets.
- Concentration Verification: Analyses of the premix, all dose levels and the 0 (control) mg/kg bw/day diets were determined prior to the start of exposure and at approximately 1.5, 3, 8, 12, 18, and 24 months. Additional concentration analyses were also conducted at approximately 14, 18, and 20 months. The method for analysing the test material in feed was a solvent extraction method followed by analyses using liquid chromatography- mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
- Retainer Samples: Reference samples (one/sex/dose/mix) were retained and stored in sealed vials in a manner consistent with the sample retention policy of the laboratory. - Duration of treatment / exposure:
- Two years (chronic groups)
Ninety days (sub-chronic groups) - Frequency of treatment:
- Daily.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- subchronic groups; Basis:
nominal in diet - Dose / conc.:
- 1 mg/kg bw/day (nominal)
- Remarks:
- subchronic groups; Basis:
nominal in diet - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- chronic groups; Basis:
nominal in diet - Dose / conc.:
- 0.1 mg/kg bw/day (nominal)
- Remarks:
- chronic groups; Basis:
nominal in diet - Dose / conc.:
- 1 mg/kg bw/day (nominal)
- Remarks:
- chronic groups; Basis:
nominal in diet - Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- chronic groups; Basis:
nominal in diet - Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- chronic groups; Basis:
nominal in diet - No. of animals per sex per dose:
- Ten per sex per dose - subchronic groups
Ten per sex per dose - chronic groups - Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Dose levels for this study were selected based on the toxicity data from a subchronic toxicity study (Yano and Dryzga, 2001), a non-guideline pharmacokinetic study (Saghir and Mendrala, 2002) and written and verbal consultations with the U.S. E.P.A.
For the chronic toxicity/oncogenicity portion of the study the targeted dose levels were 0, 0.1, 1.0, 75, and 300 mg/kg bw/day.
Based on the data from the 90-day dietary toxicity study, as well as the pharmacokinetic study, proposed dose levels were 0, 0.1, 1.0, 10, and 75 mg/kg bw/day. Data from these studies indicated that gastrointestinal absorption was nearly saturated at an actual dose of 78-86 mg/kg (targeted concentration of 100 mg/kg). Numerous toxicology data points, especially liver weights, supported this conclusion. In the 90-day dietary toxicity study, absolute and relative liver weights were increased 17 % and 21 % for males and 30 % and 34 % for females at the 100 mg/kg bw/day dose level. In subsequent discussions with the EPA, the Agency noted that the original proposed dose levels may be sufficient to produce adequate toxicity in the chronic study, but that a more conservative approach would be to use a higher dose which would provide greater assurance of safety (EPA, 2002). Therefore, the highest-dose level was selected at 300 mg/kg bw/day because additional test material was absorbed at a dose higher than 100 mg/kg bw/day, although the dose was not proportional to toxicity or the area under the curves (AUCs). As previously noted in discussions with the EPA, this reflected a conservative approach to dose level selection. Treatment-related effects induced in rats given 300 mg/kg bw/day were expected to be as follows: 1) slight reduction in body weight, 2) increased liver weights, and 3) possible alterations in clinical chemistry parameters.
Similar effects were expected to occur in rats given 75 mg/kg bw/day as would occur in rats given 300 mg/kg bw/day due to a saturation of absorption at a dose of approximately 75 mg/kg bw/day.
- Dosing route selection rationale: Potential routes of human exposure to the test material would be via ingestion during application or manufacture. Thus, administration of the test material to rats via the diet represented an appropriate means of exposure.
- Rationale for animal assignment: Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side (general) clinical examination was conducted at least once a day, typically at the same time each day (usually in the morning). Moribund animals not expected to survive until the next observation periods were humanely euthanised that day. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
- Cage side observations included, but not limited to: Decreased/increased activity, repetitive behaviour, vocalisation, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urinary quantity.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO's) were examined for the 90 day sub-chronic toxicity group only; the 2 year chronic toxicity group was not examined.
Observations during the 90 day toxicity study were recorded on the following days; 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 (day 90 in females only).
The DCO’s were conducted on animals at approximately the same time each examination day according to an established format. Categorical observations, clinical and cage-side non-scheduled observations in which only positive findings were documented are summarised collectively as clinical observations.
- Parameters checked included:
> Inside home cage: abnormal movements or behaviour, gait evaluation, and palpebral closure.
> Outside home cage: postural thrust response, responsiveness to touch, bilateral eye movement, pupil reactivity, pupil reactivity, lacrimation (clear periocular wetness), salivation (clear perioral wetness), muscle tone, visual response, and reactivity to observer.
> Categorical observations: abnormal behaviour, abnormalities of the eye, abnormal urine and faeces, abnormalities of the gastrointestinal (GI) tract, injury, missing extremity, abnormal muscle movements, palpable mass/swellings, abnormal posture, abnormalities of the reproductive tract, abnormal respiration, abnormal skin or hair-coat/mucous membranes, excessive soiling, and general abnormalities.
BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed during the pre-exposure period, weekly during the first 13 weeks of the study and then at approximately monthly intervals during the remainder of the study. Body weight gains were calculated throughout the study.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption: Feed consumption data were collected weekly during the first 13 weeks of the study and then at approximately monthly intervals thereafter for all animals. Feed containers were weighed at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed containers - final weight of feed containers) / [(No. of days in measurement cycle)(No. of animals per cage)]
- Test material intake (TMI): TMI was calculated for 0-90 days, 0-12 months and 12-24 months using test material concentrations in the feed, actual body weights and measured feed consumption in the following equation:
TMI = [(feed consumption) * (1000) * (% of test material in feed/100)] / {[(current BW + previous BW) / 2] / 1000}
FOOD EFFICIENCY:
Feed efficiency was calculated using body weight gain and feed consumption data from the first 13 weeks of the study using the following equation:
Feed efficiency = (g feed consumed/day) / (g body weight gain/day)
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to termination using indirect ophthalmoscopy.
- Dose groups that were examined: All groups were examined.
- Method: One drop of 0.5 % tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. A prosector also examined eyes during necropsy through a moistened glass slide pressed to the cornea.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus of anesthetised animals. Samples were taken from 10 rats/sex/dose from the chronic toxicity group at 3, 6, and 12 months; and the subset of animals designated for the subchronic study. The blood samples were taken after the FOB testing was completed on the neurotoxicity subset of this group.
- Anaesthetic used for blood collection: Yes (CO2).
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose group from all dosing groups.
- Haematology sample preparation: Blood samples for a complete blood count from the main group were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright’s stain and archived. Hematologic parameters were assayed using a Technicon H.1E Haematology Analyser.
- Coagulation sample preparation: Blood samples for coagulation were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000.
- Haematology parameters checked: Haematocrit (Hct), haemoglobin (Hgb) concentration, red blood cell (RBC) count, total white blood cell (WBC) count, platelet (PLAT) count, differential WBC count, and RBC indices - mean corpuscular haemoglobin concentration (MCH), mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC).
- Coagulation parameters checked: Prothrombin time (PT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the orbital sinus of anesthetised animals. Samples were taken from 10 rats/sex/dose from the chronic toxicity group at 3, 6, and 12 months; and the subset of animals designated for the subchronic study. The blood samples were taken after the FOB testing was completed on the subset of this group.
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose group from all dosing groups.
- Sample preparation: Blood samples were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyser.
- Parameters checked:
> Enzyme Activities of: Alkaline phosphatase (AP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)
> Concentrations of: Albumin (ALB), cholesterol (CHOL), creatinine (CREAT), electrolytes (Na, K, PO4, Cl and Ca), glucose (GLU), total bilirubin (TBILI), total protein (TP), and urea nitrogen (UN).
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all surviving non-fasted rats from the chronic toxicity group at 3, 6 and 12 months (Note - One control female (animal # 02A2147) from the neurotoxicity group died and was replaced with the first surviving rat from the shared chronic/neurotoxicity group (animal # 02A2141) for neuropathologic effects. Urine from female 02A2147 was collected, analysed and reported with the chronic toxicity/oncogenicity group). A urinalysis was also conducted on the subset of animals designated for the 90-day toxicity evaluation prior to necropsy. Urine was also collected by manual compression of the bladder prior to the necropsy for characterisation of the microsediment using a pooled sample from each dose group/sex.
- Metabolism cages used for collection of urine: Yes, animals were housed in metabolism cages and urine collected overnight (approximately 16 hours).
- Animals fasted: No
- Parameters checked:
> Assays: Colour, appearance, specific gravity (refractometer) and urine volume.
> Semiquantitative analysis of: pH, bilirubin, glucose, proteins, ketones, blood, and urobilinogen. - Sacrifice and pathology:
- NECROPSY
Fasted rodents, submitted alive, were anesthetised by the inhalation of CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate- buffered 10 % formalin using a hand-held syringe and blunt needle.
Similar necropsy procedures were followed for animals found dead or moribund, except that body weights, organ weights, standard blood samples (only blood smears on moribund animals), and urine samples were not obtained.
Moribund animals that were not expected to survive until the next observation period, and any animals found dead were necropsied on that day. However, animals euthanised in moribund condition or found dead after routine working hours or on weekends or holidays were refrigerated until the next scheduled work day at which time they were necropsied.
GROSS PATHOLOGY: Yes
- Organ weights: The brain, liver, kidneys, heart, adrenals, thymus (90 days, only), testes, epididymides, ovaries, uterus, and spleen.
HISTOPATHOLOGY: Yes
- Subchronic Toxicity Groups: The liver from the 0 and 1.0 mg/kg bw/day groups were processed and examined.
- Chronic Toxicity Groups: The tissues listed below were processed by standard histologic procedures from control and high-dose group animals and all animals that died or were sacrificed in a moribund condition. (Note - One control female (animal # 02A2147) from the neurotoxicity group died and was replaced with the first surviving rat from the shared chronic/neurotoxicity group (animal # 02A2141) for neuropathologic effects. Histology data from control female 02A2147 was examined and reported with the chronic toxicity/oncogenicity data sets). The following tissues from the remaining groups were processed and examined: liver, lungs, kidneys, relevant gross lesions, target organs and other selected organs.
> Tissues examined: Adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/harderian gland, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina. - Statistics:
- For details on the statistics used in this study see below in the field "Any other information on materials and methods incl. tables".
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment related effects in the first year. Treatment related clinical observations recorded during the second year of dosing; higher incidence of missing parts of the tail (males given 300 mg/kg bw/day).
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- No treatment related effects in the first year. Treatment related clinical observations recorded during the second year of dosing; higher incidence of missing parts of the tail (males given 300 mg/kg bw/day).
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Lower body weights and body weight gains (males and females given 75 and 300 mg/kg bw/day).
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related effects.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased prothrombin time (males given 75 and 300 mg/kg bw/day).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased cholesterol levels (females given 75 and 300 mg/kg bw/day); higher alkaline phosphatase activities (males and females given 75 and 300 mg/kg bw/day).
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Higher urine volume (males and females given 75 and 300 mg/kg bw/day); lower urine specific gravity (males and females given 75 and 300 mg/kg bw/day).
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment related effects observed in males and females given 75 and 300 mg/kg bw/day, see below “Results” for more details.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment related effects observed in males and females exposed to the test material, see below “Results” for more details.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased incidences of non-neoplastic effects were observed in males and females given 75 and 300 mg/kg bw/day.
- Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased incidence of neoplasms were observed in males given 300 mg/kg bw/day and females given 75 and 300 mg/kg bw/day.
- Details on results:
- CLINICAL SIGNS AND MORTALITY
- Mortality: Over the course of the study, 19, 12, 16, 16, and 14 males died spontaneously or were humanely euthanised due to their moribund condition from the 0, 0.1, 1, 75, or 300 mg/kg bw/day dose groups, respectively. For females, 9, 14, 16, 11, and 4 rats died or were humanely euthanised from the 0, 0.1, 1, 75, or 300 mg/kg bw/day groups, respectively. There were no statistically identified differences in cumulative mortality for either males or females.
- Detailed Clinical Observations: Isolated observations in males and females involving unilateral or bilateral increases in pupil size and decreased amounts of faeces were interpreted not to be treatment related due to the low incidence and lack of a dose-response relationship.
- Clinical Observations: During the first year, all observations were interpreted as not being treatment related. Four treated males (1.0 mg/kg bw/day) and six females (0.1 mg/kg bw/day) had periocular soiling, and up to four females had urine soiling (0.1 mg/kg bw/day). These findings were sporadic, transient, lacked a dose response and were interpreted not to be treatment related. Observations other than those previously discussed occurred in only one or two rats/sex/dose group and/or there was no pattern suggesting an increased occurrence in relation to exposure to the test material.
During the second year of the study, there were greater numbers and variety of observations than noted through the first year. This was an expected finding due to geriatric diseases as these rats neared the end of their life span. Increasing numbers of rats had cloudy eyes, preputial or clitoral gland swelling, papules/pustules of the skin, soiling - urine and periocular, and missing part of the tail. Papules/pustules of the skin (generalised distribution) affected the tail and were higher in incidence than the controls for males given 0.1, 1.0, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day. The increased incidence of papules/pustules of the skin of males given 0.1 and 1.0 mg/kg bw/day were not dose related and were interpreted not to be treatment related. Males given 300 mg/kg bw/day also had a higher incidence of missing part of the tail than the control or other dosed rats. The interpretation of these differences is discussed in the histopathology section (below).
Cloudy eyes (males and females), preputial/clitoral gland swellings and soiling (urine and periocular) were not dose related and were interpreted not related to the test material. Urine soiling was observed more often in females given 75 and 300 mg/kg bw/day around day 568; however, the incidence decreased markedly by the end of the study, suggesting that these nonspecific observations were not toxicologically significant. All other in-life observations not previously discussed were also interpreted not to be treatment related.
Masses were infrequently found on the categorical portion of DCO examinations through the first 12 months of study, with only one male given 300 mg/kg bw/day having a small mass on the nose that later resolved. As the study progressed, the incidence of palpable masses increased; however, there was no evidence of a dose-response relationship in the incidence of palpable masses. The resolution of palpable masses is addressed following the gross and histopathologic examination of these lesions.
BODY WEIGHT AND WEIGHT GAIN
Body weights for the male rats given 75 or 300 mg/kg bw/day were lower than controls and were interpreted to be treatment related. The differences from controls were first statistically identified at day 85, when the males given 75 or 300 mg/kg/day weighed 2.5 % and 2.7 % less than controls, respectively. Body weights of these males remained lower than the controls throughout the study and were frequently statistically identified as different from the controls. The body weights of males given 300 mg/kg bw/day were 3.7 % lower than controls on day 363 and males given 75 mg/kg bw/day weighed 3.5 % less on day 363. Males given 75 or 300 mg/kg bw/day weighed 7.0 and 9.7 % less than controls, respectively, on day 729, when the in- life phase of the study was complete. Body weights of male rats given 1.0 mg/kg bw/day were slightly lower than controls throughout the study; however, the differences were not attributed to treatment, as they were generally within 2 % of the control body weights. Body weights of males given 0.1 mg/kg bw/day were comparable to the controls throughout the study.
Body weights for the females given 75 or 300 mg/kg bw/day were lower than controls and were treatment related. Decreases from controls were first statistically identified on day 176, when the females given 300 mg/kg bw/day weighed 2.5 % less than controls. Body weights for females given 75 mg/kg bw/day were statistically identified and 3.2 % lower than controls on day 316. Body weights for the 75 and 300 mg/kg bw/day groups continued to decrease and were statistically identified for the remainder of the study. Body weights of female rats given 75 or 300 mg/kg bw/day were 4.0 % and 6.0 % lower than controls on day 363, and were 14.0 % and 14.7 % less than controls, respectively on day 729. Body weights for female rats given 0.1 or 1.0 mg/kg bw/day were comparable to controls throughout the study.
Differences in body weights were reflected in lower body weight gains for males and females given 75 or 300 mg/kg bw/day.
Body weight gains of males given 75 or 300 mg/kg bw/day were 4.6 % and 5.1 % less than controls, respectively on day 363. These decreases remained until study termination, at which time they were 9.5 % and 13.9 % lower than controls for the 75 and 300 mg/kg bw/day groups, respectively. Body weight gains for females given 75 or 300 mg/kg bw/day were 6.5 % and 9.8 % lower than controls on day 363 and 21.8 % and 22.4 % lower than controls at study termination. The lower body weight gains of males given 300 mg/kg bw/day and females given 75 or 300 mg/kg bw/day clearly exceed the 10 % body weight gain decrement criteria, as defined by several regulatory authorities (EPA, 2003; HED, 2003; Commission Directive, 2001), and males given 75 mg/kg bw/day almost meet the 10 % criteria and may confound the interpretation of tumorous effects at these dose levels.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed Consumption: Feed consumption values for males given 1 or 300 mg/kg bw/day were higher than controls (< 6 %) and frequently statistically identified through day 407; however, from day 407 to study termination, feed consumption for males given 300 mg/kg bw/day were comparable to controls at most time points. In contrast, feed consumption for males given 1.0 mg/kg bw/day continued to be higher than the controls and statistically identified and was 8.4 % higher at study termination. Feed consumption for males given 0.1 or 75 mg/kg bw/day was considered comparable to controls throughout the study. The cause of the slightly higher feed consumption observed through day 407 for males given 300 mg/kg bw/day was not determined.
Feed consumption values for females given 1.0, 75, or 300 mg/kg bw/day were higher (< 7 %) than controls from day 1-379 and frequently statistically identified. After day 379, feed consumption values of females given 75 or 300 mg/kg bw/day were lower than controls and statistically identified. In contrast, feed consumption values of females given 1.0 mg/kg bw/day, continued to be higher than the controls and statistically identified throughout the remainder of the study. The cause of the feed consumption variability observed for females given 75 or 300 mg/kg bw/day was not determined. Feed consumption of females given 0.1 mg/kg bw/day was comparable to the controls.
These apparent differences in feed consumption may reflect normal biologic variability between groups and were interpreted not to be treatment related. The apparent increase in feed consumption observed at 1.0 mg/kg bw/day was considered unrelated to treatment because of a lack of a dose-response relationship.
- Test Material Intake: The test material intakes were very close to the targeted dose for all levels over the course of the study. The time weighted average amounts of ingested test material (mg/kg bw/day) were within 5 % of the targeted dosages for all dose groups.
Months 0-12: The actual doses received were 0.100 (± 0.006), 1.00 (± 0.06), 75.3 (± 4.2) and 303 (± 17) mg/kg bw/day for males and 0.099 (± 0.005), 0.99 (± 0.07), 74.5 (± 3.3) and 298 (± 14) for females dosed at 0.1, 1.0, 75 and 300 mg/kg bw/day, respectively.
Months 0-24: The actual doses received were 0.101 (± 0.006), 1.01 (± 0.06), 78.7 (± 13.4) and 303 (± 15) mg/kg bw/day for males and 0.101 (± 0.005), 1.01 (± 0.06), 75.5 (± 3.0) and 303 (± 13) for females dosed at 0.1, 1.0, 75 and 300 mg/kg bw/day, respectively.
FOOD EFFICIENCY
Feed efficiency between the control and treated males and females were generally comparable during most time points. On occasion there were instances of apparent increased or decreased feed efficiency. These differences were not consistently dose related and were interpreted not to be treatment related.
OPHTHALMOSCOPIC EXAMINATION
Pre-exposure examination of rats placed on study indicated all rats were within normal limits. Variable numbers of male and female rats had a pale fundus, cloudy cornea, periocular soiling, opaque cornea, opaque lens, phthisis bulbi, incomplete pupil dilatation, or missing, enlarged eye or microphthalmia at the 12- and 24- month intervals. Periocular soiling was considered to be a non-specific observation, while eyes with pale fundus, opaque cornea, opaque lens, or cloudy cornea were interpreted to be spontaneous, age-related changes.
HAEMATOLOGY
- Treatment Related Effects
Prothrombin time was consistently elevated in males given 75 or 300 mg/kg bw/day and was interpreted to be treatment related. Males given 300 mg/kg bw/day had statistically identified elevations in prothrombin time beginning at 3 months, while in males given 75 mg/kg bw/day, this occurred at 6 months. The elevations in prothrombin time were evident until 18 months in both of these treatment groups. At no time during the study were there clinical observations of haemorrhage or bruising that could reflect an adverse effect of this slightly longer prothrombin time.
Prothrombin times at 24 months in males given 75 or 300 mg/kg bw/day were similar to the controls. The lack of a difference in prothrombin time between males given 0 mg/kg bw/day and those given 75 or 300 mg/kg bw/day at 24 months may be due to increased variability in prothrombin times associated with confounding disease processes in rats of this age.
- Effects Not Related To Treatment
Prothrombin times in females were slightly lower than the controls and statistically identified at 18 months in the 75 and 300 mg/kg bw/day dose groups. These differences were not observed at 3, 6, 12, or 24 months, and were interpreted not to be treatment related. Moreover, slight decreases in prothrombin time would not normally be considered toxicologically significant.
Males given 75 or 300 mg/kg bw/day had haematocrits that were slightly higher than the controls at 18 and 24 months and these differences were statistically identified. However, these values were within the historical control range. Males given 75 or 300 mg/kg bw/day also had statistically identified higher RBC counts observed at these time points. The MCV values (ratio of the haematocrit to RBC count), were slightly lower in males given 75 or 300 mg/kg bw/day at 18 and 24 months, relative to the concurrent controls; however, they were close to the historical control values and therefore interpreted not to be treatment related. Moreover, the mean concurrent control MCV (61.2) of males at 24 months was unusually high compared to historical controls, further suggesting that the alterations in MCV were not treatment related.
Additionally, there were no treatment-related changes in the bone marrow suggestive of increased output of red blood cells. Haemoglobin values were also similarly elevated and statistically identified in males given 75 or 300 mg/kg bw/day at 18 and 24 months. Haemoglobin values were within or in close proximity to historical control values. In addition, the concurrent controls were outside the range (lower) of historical controls and the increases in haemoglobin values were not physiologically or toxicologically significant. Therefore, the increased haematocrit, increased RBC counts and increased haemoglobin values observed at the 18 and 24 months in males given 75 or 300 mg/kg bw/day were interpreted not to be treatment related.
Statistically identified changes that were considered spurious included slightly lower values in the 12- month haematocrit and haemoglobin of females given 300 mg/kg bw/day, a slightly lower value in the 18- month haemoglobin concentration of females given 75 mg/kg bw/day and a slight increase in the 6- month haemoglobin concentration of males given 75 mg/kg bw/day. Similarly, higher platelet counts in males given 300 mg/kg bw/day at 3 and 6 months, and females given 75 or 300 mg/kg bw/day at 6 months were also considered spurious. These statistically identified changes were considered not toxicologically relevant due to lack of consistency across time points and/or lack of dose responsiveness.
CLINICAL CHEMISTRY
- Treatment Related Effects
Females given 75 or 300 mg/kg bw/day had slightly higher cholesterol (CHOL) levels than the controls throughout the study. These differences in CHOL levels were higher than the historical control ranges and were interpreted to be treatment related, but were not associated with a clear adverse effect.
Males and females given 75 or 300 mg/kg bw/day had higher alkaline phosphatase activities (ALP) than the controls throughout the study, were frequently statistically identified and included: males and females given 75 mg/kg bw/day at 3, 6, 12, and 18 months, and males and females given 300 mg/kg bw/day at 3, 6, 12 (males only), and 18 (females only) months. These differences were higher than the concurrent controls, generally were outside of the historical control ranges, associated with higher liver weights and hepatocellular hypertrophy, and were interpreted to be treatment related.
- Effects Not Related To Treatment
Males given 75 or 300 mg/kg bw/day had higher CHOL levels than the controls at selected times during the study, which were frequently statistically identified, and included: 75 mg/kg bw/day at 3 months, and 300 mg/kg bw/day at 3 and 12 months. The CHOL differences in males were not observed at 18 and 24 months and were not attributed to the test material.
Males given 75 or 300 mg/kg bw/day had higher total protein (TP) than the controls throughout the study, which were frequently statistically identified and included: 75 mg/kg bw/day at 3, 6, 12, 18, and 24 months and males given 300 mg/kg bw/day at 3, 6, and 12 months. These differences were higher than the concurrent controls but in close proximity to the high end of the historical control ranges and were interpreted not to be treatment related.
Males given the test material had higher urea nitrogen (UN) concentrations throughout the study, were frequently statistically identified and included: 1.0 mg/kg bw/day at 3 months, 75 mg/kg bw/day at 3, 6, 12, and 18 months, and 300 mg/kg bw/day at 3, 6, 12, 18, and 24 months. In general, these differences were only slightly higher than the concurrent controls and frequently within the historical control ranges. Creatinine (CREAT) values of males given the test material were similar to the controls throughout the study, although the values for two dose levels (0.1 and 300 mg/kg bw/day) at 3 months were statistically different from the controls. These differences were interpreted not to be treatment related, because of a lack of dose response, the failure for the effect to occur with prolonged exposure, and the absence of a significant degree of renal disease that would correlate with these slightly higher values.
Females given 75 or 300 mg/kg bw/day also had higher UN values than the controls at selected times during the study, were frequently statistically identified and included: 75 and 300 mg/kg bw/day at 6 and 18 months. These differences were only slightly higher than the concurrent controls and/or frequently within the historical control ranges and were not attributed to the test material. Creatinine (CREAT) values of females given the test material were similar to the controls throughout the study, not statistically identified as different and were not indicative of kidney disease.
Males given 75 or 300 mg/kg bw/day had lower potassium concentration than the controls at 3 months and were statistically identified. These differences were lower than the historical control range; however, similar differences were not observed at the longer exposure periods (6-24 months) and were interpreted not to be treatment related.
Males given 75 or 300 mg/kg bw/day had higher albumin levels than the controls at 3 months and were statistically identified. These differences were higher than the concurrent controls but were within the historical control range, similar differences were not observed at the longer exposure periods (6-24 months), and were therefore interpreted not to be treatment related.
URINALYSIS
- Treatment-Related Effects
Males given 75 or 300 mg/kg bw/day had higher urine volume than the controls throughout the study, were occasionally statistically identified and included: 75 mg/kg bw/day at 3, 6 (statistically identified), 12, and 18 months, and 300 mg/kg bw/day at 3, 6 (statistically identified), 12, 18 (statistically identified), and 24 months. There were a few instances when males given 1.0 mg/kg bw/day had increased urine volumes as compared to the controls. The differences in males given 300 mg/kg bw/day were almost always higher than the historical control ranges and were interpreted to be treatment related. Differences in males given 1.0 or 75 mg/kg bw/day were not consistently elevated above concurrent or historical control values and were not attributed to treatment with the test material.
Females given 75 or 300 mg/kg bw/day had higher urine volume than the controls throughout the study, were frequently statistically identified and included: 75 mg/kg bw/day at 3, 6, 12, 18, and 24 months, and 300 mg/kg bw/day at 3, 6, 12, 18, and 24 months. There were also a few instances when females given 1.0 mg/kg/day had increased urine volumes.
The urine volume differences in females given 75 or 300 mg/kg bw/day were attributed to treatment with the test material because the values were outside of the concurrent and historical control values. Urine volumes of females given 1.0 mg/kg bw/day were interpreted to be within normal limits.
Males and females given 75 or 300 mg/kg bw/day had lower urine specific gravity than the controls throughout the study, were frequently statistically identified and included: 75 mg/kg bw/day at 3 (males and females), 6 (males and females), 12 (males and females), 18 (males and females) and 24 (females) months, and 300 mg/kg bw/day at 3 (males and females), 6 (males and females), 12 (males and females), 18 (males and females) and 24 (males and females) months. In addition, there were a few instances when males and females given 0.1 or 1.0 mg/kg bw/day had lower urine specific gravity.
The differences in males and females given 75 or 300 mg/kg bw/day were almost always lower than the historical control ranges and were interpreted to be treatment related. The pathogenesis of the increased urine volume and decreased specific gravity was not clear as these rats did not have a toxicologically significant degree of renal disease and the UN and CREAT concentrations were unaffected by the test material.
- Effects Not Related To Treatment
Males given 75 or 300 mg/kg bw/day had a higher incidence of a more alkaline urine pH at all time points throughout the study, as compared to the concurrent controls. Differences were most apparent during the first 18 months of the study and were only marginally different at the end of the study.
Male urine pH data from three recently completed studies indicated that the urine pH of males given 75 or 300 mg/kg bw/day were outside of the historical control data at 3 months, but were within or in close proximity to the historical control values at 6, 12, 18, and 24 months. Based on these data, the differences in urine pH observed in this study were interpreted not to be treatment related.
Urine pH of females given the test material was similar to the concurrent controls throughout the study, with the exception of the 300 mg/kg bw/day group at 18 months. However, urine pH values at this time were similar to the historical control data. Therefore, the isolated occurrence of alkaline (pH 7) urine was not attributed to treatment to the test material.
ORGAN WEIGHTS
- Primary Treatment Related Effects
Final body weights of males given 75 or 300 mg/kg bw/day for 12 and 24 months were lower than the controls, statistically identified (24 months), and consistent with lower body weights of these males during the in-life phase of the study. Males given 75 or 300 mg/kg bw/day for 12 and 24 months had higher absolute and relative weights of the liver and kidneys. These differences were dose related, statistically identified, outside of the historical control values and were interpreted to be treatment related. Males given 75 or 300 mg/kg bw/day also had higher absolute and relative testes weights and lower absolute and relative epididymal weights at 24 months. The testes had not been previously identified to be a target organ of the test material; however, the testes weights were clearly outside of the historical control range and the differences in weight were attributed to the occurrence of larger interstitial cell tumors in males given 75 or 300 mg/kg bw/day, with no difference in the incidence of these tumors at these dose levels (see Histopathology section). Lower epididymal weights were interpreted to be a secondary effect of the larger testicular neoplasms, resulting in a greater degree of seminiferous tubule atrophy and ultimately less spermatic elements in the epididymides.
Females given 75 or 300 mg/kg bw/day for 12 and 24 months also had higher absolute and relative weights of the liver and adrenals. These differences in liver weight were dose related, whereas the adrenal weights were similar. These organ weights were statistically identified, outside of the historical control values and were interpreted to be treatment related.
- Secondary Treatment Related Effects
Final body weights of males given 75 or 300 mg/kg bw/day for 12 and 24 months were lower than the controls, statistically identified (24 months), and consistent with lower body weights of these males during the in-life phase of the study. These lower body weights were associated with higher relative weights of the heart (12 and 24 months) and brain (24 months). Males given 75 or 300 mg/kg bw/day at 24 months also had lower absolute and relative spleen weights than the concurrent or historical controls. The lower spleen weight of these males was attributed to a lower incidence of Fischer 344 rat leukemia.
Final body weights of females given 75 or 300 mg/kg bw/day for 12 and 24 months were a) lower than the controls, b) statistically identified (24 months), c) were consistent with lower body weights of these females during the in-life phase of the study, and d) were interpreted to be treatment related. These lower body weights were associated with higher relative weights of the heart (12 and 24 months), kidneys (12 and 24 months), and brain (24 months). Females given 300 mg/kg bw/day had a higher relative spleen weight (12 months), and females given 75 or 300 mg/kg bw/day had lower absolute and relative spleen weights at 24 months. The lower spleen weights of these females at 24 months were attributed to a lower incidence of Fischer 344 rat leukemia.
- Effects Not Related to Treatment
The weights of the adrenal glands (absolute – males given 300 mg/kg bw/day; relative – males given 75 or 300 mg/kg bw/day) at 24 months were higher than the concurrent control values and were interpreted not to be treatment related because the values were within the historical control range.
Females given 75 or 300 mg/kg bw/day for 24 months had lower absolute and relative uterine weights than the concurrent controls. Uterine weights were similarly interpreted not to be treatment related because the values were within or in close proximity to the historical control values and the concurrent values appeared to be an outlier, since those values were outside of the historical controls.
GROSS PATHOLOGY
> 12 Months:
- Treatment-Related Effects
The presence of foci on the surface of the lungs in the majority of males and almost all females given 300 or 75 mg/kg bw/day was interpreted to be treatment related. These foci were pale, focal or multifocal, either confined to one lobe or noted in multiple lobes. Such foci were seen on the lungs of 6 of 10 males given 75 or 300 mg/kg bw/day and in 8 of 10 and 10 of 10 in females given 75 or 300 mg/kg bw/day, respectively. These foci on the lungs represented small areas of chronic inflammation (see Histopathology section). One male given 300 mg/kg bw/day for 12 months had a mass nodule on the liver that was diagnosed as an adenoma (liver adenomas were interpreted to be treatment related, see Histopathology section). This mass nodule on the liver was interpreted to be treatment related because liver tumors were not observed at 12 months in three previous chronic toxicity/oncogenicity studies and because of the significant increase in liver tumors seen at 24 months at this dose level.
- Effects Not Related To Treatment
The right optic nerves in some animals across all dose groups and in both sexes were either decreased in size or missing. This finding was not associated with the exposure to the test material and was attributed to the technical artifact of repeated retro-orbital bleeding at 3-, 6-, and 12-months. Grossly observed mass nodules unrelated to treatment consisted of 3, 1, 0, and 1 uterine polyp(s) in females given 0.1, 1.0, 75, or 300 mg/kg bw/day, respectively. The low incidence and lack of dose response indicated that the uterine polyps were not treatment related at 12 months. Additionally, one control female (animal #02A2147) designated for the 12- month necropsy that was euthanised on day 232 due its moribund condition, had mass nodules in the mesenteric and mediastinal tissues. Data from this rat were incorporated in the gross and histopathology data from the 12-month necropsy. Moribundity in this animal was attributed to the presence of the mass nodule in the mesenteric tissue, which was diagnosed histologically to be an invasive and malignant rhabdomyosarcoma of the skeletal muscle of the diaphragm. All other gross observations were considered spontaneous changes unrelated to treatment, commonly found in Fischer 344 rats of this age and husbandry conditions.
> 24 Months:
- Treatment-Related Effects
There was an increased incidence of one or more foci on the surface of the lungs in majority of males and females given 75 or 300 mg/kg bw/day for up to 24 months. This was similar to what was observed at 12 months. Foci were pale, focal, or multifocal; either confined to one lobe or noted in multiple lobes. They represented small areas of chronic inflammation (see Histopathology section below). In the liver, there was a higher incidence of small foci (pale/dark/focal/multifocal) on one or more lobes of the liver in males given 300 mg/kg bw/day. In general, many of these grossly observed foci represented eosinophilic and/or basophilic foci of cellular (hepatocyte) alteration and occasional hepatocellular adenomas (see Histopathology section for resolution of these lesions).
Females given 75 or 300 mg/kg bw/day had increased incidence of mass nodules of the uterus, particularly those that were diagnosed as stromal polyps (see Histopathology section below). The tails of males given 0.1, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day had an increased incidence of lesions diagnosed as papules (focal and multifocal). The incidences in males given 0, 0.1, 1.0, 75, and 300 mg/kg/day were 5,14, 7, 21, and 25, respectively and in females were 1, 1, 1, 0, and 8, respectively. These lesions generally represented focal hyperkeratosis with or without inflammation (see Histopathology section below). Additionally, males given 300 mg/kg bw/day (9/50) also had necrosis of the tail tip. Females given 300 mg/kg bw/day appeared to have a higher incidence of uterine mass/nodules – polypoid, 0.2 – 0.5 cm than the controls. These mass/nodules were subsequently interpreted to be treatment related following histopathology (see Histopathology section below).
- Effects Not Related to Treatment
The right optic nerve of females given 300 mg/kg bw/day showed a slightly higher incidence (6 vs. 1 control) of decreased size as compared to the controls. This lesion in the females was not considered treatment related because of its unilateral distribution (see Histopathology section below).
Numerous other gross observations, including several mass-nodules were noted at 24 months; however, none of these gross observations were considered indicative of treatment-related gross effects and were generally consistent with expected geriatric conditions of Fischer 344 rats of this age. - Key result
- Dose descriptor:
- NOEL
- Remarks:
- (90 days)
- Effect level:
- 1 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No treatment related observations in animals dosed at this level.
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- (1 year)
- Effect level:
- 1 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No treatment related observations in animals dosed at this level.
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of the test, the 90 day NOEL for subchronic toxicity and the 12 and 24 months NOELs for chronic toxicity was determined to be 1.0 mg/kg bw/day for both male and female Fischer 344 rats.
- Executive summary:
The subchronic and chronic toxicity of the test material was assessed in a 2 year dietary toxicity study using Fischer 344 rats. The study was performed under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 87.4300, EPA OPPTS 870.3100, OECD 453, OECD 408, EU Method Part B, EU Method B.26, and JMAFF guidelines.
Groups of 65 males and 65 female Fischer 344 rats were fed diets formulated to provide 0, 0.1, 1.0, 75, or 300 mg/kg bw/day for up to two years. An additional 10 rats/sex were given 0 or 1.0 mg/kg bw/day for 90 days to evaluate subchronic toxicity. Ten rats/sex/dose were necropsied after one year (chronic toxicity group), five rats/sex/dose were necropsied after one year (chronic neurotoxicity group) and the remaining 50 rats/sex/dose were fed the respective diets for up to two years and necropsied (oncogenicity group).
Daily cage-side examinations, weekly detailed clinical observations, and periodic body weight and feed consumption were monitored throughout the study on all rats.
Haematology, clinical chemistry and urinalysis parameters were evaluated at regular intervals throughout the study. Ophthalmic examinations were conducted on all rats pre-exposure and all surviving rats prior to the scheduled necropsies. All rats had a complete necropsy and organ weights were obtained from all rats at the scheduled necropsies. Histopathologic examinations of an extensive set of tissues were performed on all control and high-dose level rats and all rats that died spontaneously or were euthanised due to their moribund condition. Histopathologic examination of the surviving animals from the low- and intermediate-dose levels was limited to target organs, suspected target organs, and gross lesions.
Subchronic Toxicity:There were no treatment-related alterations observed in any parameter for male or females given 1.0 mg/kg bw/day, establishing this as a no-observed-effect level (NOEL) at 90 days.
Chronic Toxicity:Treatment-related effects consisted of: decreased body weight and body weight gains (males and females given 75 or 300 mg/kg bw/day), increased liver weights (males and females given 75 or 300 mg/kg bw/day), increased kidney weights (males given 75 or 300 mg/kg bw/day), and increased adrenal weights (females given 75 or 300 mg/kg bw/day). Treatment-related increases in the incidences of hepatocellular hypertrophy (males only) and lung inflammation were also observed in males and females given 75 or 300 mg/kg bw/day. Two males given 300 mg/kg bw/day also had hepatocellular adenomas that were treatment related. The NOEL was 1.0 mg/kg bw/day for both males and females at 12 months.
Chronic Toxicity/Oncogenicity:Males given 300 mg/kg/day and female s given 75 or 300 mg/kg/day had increased incidences of liver adenomas and uterine stromal polyps, respectively, that were attributed to test material administration. Lower incidences of leukaemia occurred in males and females dosed with 75 or 300 mg/kg/day, compared to the concurrent controls. Males given 75 or 300 mg/kg/day had apparently larger testicular interstitial cell (Leydig cell) tumours than the controls, based on higher testicular weights; however, the incidences of interstitial cell tumours were similar at all dose levels, including the controls. Increased incidences of neoplasms were not observed in males given ≤ 75 mg/kg/day or females given ≤ 1.0 mg/kg/day.
Males and females given 75 or 300 mg/kg/day had treatment-related decreases in body weight and body weight gains, and increased liver and kidney (males only) weights. Other treatment-related effects in males and females given 75 or 300 mg/kg/day consisted of increases in the incidence of
1) lung inflammation, and
2) hyperkeratosis of the tail (300 mg/kg/day only). Males given 75 or 300 mg/kg/day had increased incidence of mineralization of renal pelvic epithelium and increased incidence of basophilic liver foci.
Males given 300 mg/kg/day also had increased incidence of hyperplasia of renal pelvic epithelium, and necrosis of the tail tip and secondary increases in the incidences of testicular seminiferous tubule atrophy and epididymal aspermia. Females given 75 or 300 mg/kg/day had a decreased incidence of basophilic liver foci. There were also minor treatment-related alterations in clinical pathology parameters in males and/or females given 75 or 300 mg/kg/day in prothrombin time, cholesterol, alkaline phosphatase activity, urine volume, and urine specific gravity. Treatment-related non-neoplastic effects were not observed in males or females given ≤ 1.0 mg/kg/day.
The no-observed-effect level (NOEL) for both males and females at 24 months was 1.0 mg/kg/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 25 September 2001 - 24 December 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- This study was not conducted to a standardised guideline as its purpose was to act as a probe in order to allow the setting of dose levels for a subsequent study.
The test material was fed in the diet to groups of eight male and eight female rats at concentrations supplying 0, 10, 100, 500 or 1000 mg/kg/day for 13 weeks to evaluate its potential toxicity. Data from this study were used to establish dose levels for a subsequent two-generation reproduction study. Parameters evaluated included daily cage-side observations, weekly clinical examinations, one detailed clinical observation, body weights, feed consumption, selected organ weights, gross and histopathologic examinations. - GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl: CD(SD) IGS BR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: Mean body weight 2 days prior to test initiation: males 170.4 g; females 134.3 g
- Fasting period before study: No
- Housing: Animals were housed one per cage in stainless steel cages with wire-mesh floors suspended above catch pans. Cages contained feed containers and pressure activated, nipple-type watering systems.
- Diet: ad libitum
- Water: municipal water provided ad libitum
- Acclimation period: Approximately 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 22.4 °C
- Humidity: 47 - 52 % (relative)
- Air changes: Approximately 12 - 15 times/hour
- Photoperiod: A 12-hour light/dark photocycle was maintained with lights on at 06:00 and off at 18:00 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared weekly based upon the most recent body weight and feed consumption data.
- Mixing appropriate amounts with (Type of food): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes were mixed periodically throughout the study based on stability data. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Continuous in the diet, 7 days per week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Basis:
nominal in diet - No. of animals per sex per dose:
- 8 animals per sex per dose
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: The high-dose level was chosen based on the results from previous 4- and 13-week studies and is a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3800 Reproduction and Fertility Effects). The intermediate- and low-dose levels provided dose response data for treatment-related effects observed in the high-dose group.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, a visual evaluation was made for morbidity, moribundity, mortality, and the availability of feed and water. To the extent possible alterations in faecal and urinary output, skin, fur, mucous membranes, respiration, central nervous system function and animal behaviour were recorded.
Once weekly, a more detailed clinical examination was conducted. In addition to the normal daily observation described above, this examination included a hands-on evaluation for swellings and/or masses. Swellings or masses observed in-life were evaluated during necropsy with a final interpretation based upon histopathologic findings, if deemed necessary, for a definitive diagnosis. The clinical examination may have replaced one of the daily observations on the day that it was conducted
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were subjected to detailed clinical observations (DCO) on test day 88 to evaluate their clinical status in more detail. DCO were conducted on all animals. The examination included cage-side, hand-held and open field observations that were recorded categorically or using explicitly defined scales (scored).
BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and weekly during the exposure period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumed was determined during the pre-exposure period and weekly during the exposure period for all animals by weighing feed crocks at the start and end of a measurement cycle. Consumption was calculated using the following formulation:
Feed consumption (g/day) = (initial weight of feed crock - final weight of feed crock) / # of days in measurement cycle
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- SACRIFICE
Fasted rodents submitted alive for necropsy were anaesthetised by the inhalation of carbon dioxide and weighed. Their tracheas were exposed and clamped, and the animals were euthanised by decapitation.
GROSS PATHOLOGY
A complete necropsy was conducted on all animals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle.
The liver, kidneys, adrenal glands, testes, and epididymides were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.
Representative samples of the following tissues were collected and preserved in neutral, phosphate-buffered 10% formalin: adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve – optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland - females only, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues nasal tissues, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve -tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina.
HISTOPATHOLOGY
Histologic examination of the adrenal glands, brain, epididymides, testes, liver and relevant gross lesions were conducted on all control and high-dose animals as listed above. Examination of tissues from the remaining dose groups were limited to tissues that demonstrated treatment-related histologic effects at the high-dose level (adrenal glands, epididymides and liver) and relevant gross lesions. Eight cross-sections of the brain from all rats in the control and high-dose groups were prepared from: cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, pons, cerebellum, and medulla oblongata. Tissues were processed by standard histologic procedures, embedded in paraffin, sectioned approximately 6 μm thick and stained with haematoxylin and eosin. Tissues were evaluated using a light microscope.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25 % involvement of the parenchyma. This type of change was not expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions of sufficient severity and/or extent (up to 50 % of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally, lesions graded as moderate were not life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life threatening. - Statistics:
- DCO incidence scores were statistically analysed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Body weights, feed consumption, and organ weights (absolute and relative) were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Both the Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experiment-wise alpha level. Feed consumption values were excluded from analysis if the feed had been spilled or scratched. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and were routinely excluded from feed consumption only. Other outliers were only excluded from analysis for documented, scientifically sound reasons.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data took into consideration statistical analyses along with other factors such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- see below
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- see below
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Examinations performed on all animals prior to the study revealed no significant findings. All animals survived to the scheduled necropsy.
On test day 71, two females (one in the 100 mg/kg/day group and one in the 1000 mg/kg/day group) exhibited single episodes of convulsions. During subsequent routine observations, four additional treated animals exhibited episodes of convulsions, including one female given 500 mg/kg/day and two males and one female given 1000 mg/kg/day. One animal had a convulsion on more than one occasion. Excess salivation, vocalisation, increased activity and attempts to bite were noted in one or more of the animals that convulsed. All rats were subjected to DCO on test day 88 to evaluate their clinical status in more detail. During DCO examination, one high-dose female had convulsions and excess salivation. Two males in the 100 mg/kg/day dose group exhibited a variety of clinical signs, including avoidance to approach, attempts to bite, and increased activity and reactivity. Both animals had increased resistance to removal and increased responsiveness to touch. One of these rats had increased reactivity to stimuli and had convulsions later the same day following the DCO. No other significant observations were recorded during the DCO examinations. Thus, a total of eight animals experienced convulsions over the course of this study. Treatment-related convulsions were not observed in previous toxicity studies using either Fischer 344 rats or CD-1 mice, nor were they observed in CD rats dosed with up to 1000 mg/kg/day of the test material for 15 days by oral gavage in a developmental toxicity study.
Recently, in three current studies using rats originating from the same supplier, convulsions were observed in three animals, two of which were control rats. These convulsions were not treatment-related and suggest a genetic predisposition to the convulsive episodes. Convulsions have not been observed previously in CD rats in the testing laboratory. At this time, it is not known whether the test material caused convulsions in the present study, contributed to the onset of convulsions in rats with a genetic predisposition for this condition, or whether the convulsions were a spurious finding due to a genetic anomaly in these animals and were unrelated to the test material.
BODY WEIGHT
There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. However, males given 100, 500 or 1000 mg/kg/day exhibited slight, non-significant decreases in terminal body weight of 4, 5 and 7 %, respectively.
FEED CONSUMPTION
There were no significant differences in the amount of feed consumed by any treated group when compared to their respective controls.
GROSS PATHOLOGY
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.
ORGAN WEIGHTS
Treatment-related alterations in organ weights are presented in Table 1. Liver and adrenal gland weights of male and female rats given 100, 500, or 1000 mg/kg/day were increased and statistically identified (with the exception of adrenal gland weights of males given 500 mg/kg/day). In addition, epididymal weights of male rats given 100, 500, or 1000 mg/kg/day were decreased and statistically identified. These organ weight changes plateaued at doses ≥ 100 mg/kg/day.
There were no significant changes in organ weights in the 10 mg/kg/day dose group.
HISTOPATHOLOGY
Selected histopathologic observations are summarised in Table 2. Histological examination revealed microscopic liver effects consisting of centrilobular hypertrophy of hepatocytes in males and females given ≥ 100 mg/kg/day, and vacuolisation consistent with fatty change in periportal hepatocytes of males given ≥ 100 mg/kg/day and females given ≥ 10 mg/kg/day. A microscopic adrenal gland effect consisted of hypertrophy of the zona fasciculata in males given ≥ 100 mg/kg/day and females given ≥ 10 mg/kg/day. A microscopic effect of the epididymides consisted of decreased spermatic elements, characterised by a very slight decrease in the amount of normal sperm present in the tail of the epididymides, in males given ≥ 100 mg/kg/day. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 10 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Dose descriptor:
- other: No NOEL determined
- Sex:
- female
- Remarks on result:
- not measured/tested
- Remarks:
- Effect level not specified (migrated information)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, the no-observed-effect level (NOEL) for males was 10 mg/kg/day. The NOEL for females was not determined.
- Executive summary:
The sub-chronic repeated dose toxicity of the test material was investigated in the Crl: CD(SD) IGS BR strain of rats under GLP conditions. The study was not conducted to a standardised guideline as its purpose was to act as a probe in order to allow the setting of dose levels for a subsequent study.
The test material was fed in the diet to groups of eight male and eight female rats at concentrations supplying 0, 10, 100, 500 or 1000 mg/kg/day for 13 weeks to evaluate its potential toxicity. Data from this study were used to establish dose levels for a subsequent two-generation reproductive toxicity study. Parameters evaluated included daily cage-side observations, weekly clinical examinations, one detailed clinical observation, body weights, feed consumption, selected organ weights, gross and histopathologic examinations.
Liver and adrenal gland weights of male and female rats given ≥ 100 mg/kg/day were increased and statistically identified (with the exception of adrenal gland weights of males given 500 mg/kg/day). In addition, epididymal weights of male rats given ≥ 100 mg/kg/day were decreased and statistically identified. These organ weight changes plateaued at doses ≥ 100 mg/kg/day. Histological examination revealed microscopic liver effects consisting of centrilobular hypertrophy of hepatocytes in males and females given ≥ 100 mg/kg/day, and vacuolisation consistent with fatty change in periportal hepatocytes of males given ≥ 100 mg/kg/day and females given ≥ 10 mg/kg/day. A microscopic adrenal gland effect consisted of hypertrophy of the zona fasciculata in males given ≥ 100 mg/kg/day and females given ≥ 10 mg/kg/day. A microscopic effect of the epididymides consisted of decreased spermatic elements, characterised by a very slight decrease in the amount of normal sperm present in the cauda epididymides, in males given ≥ 100 mg/kg/day.
Eight treated animals exhibited isolated episodes of convulsions, including one male and one female given 100 mg/kg/day, one female given 500 mg/kg/day and two males and three females given 1000 mg/kg/day. Only two animals had convulsions on more than one occasion. Excess salivation, vocalisation, increased activity and attempts to bite were also noted in one or more of the animals that convulsed. There were no microscopic effects on the brain of any high-dose animals. In addition, treatment-related convulsions were not observed in previous toxicity studies using either Fischer 344 or CD rats or CD-1 mice. However, in three current studies at the testing facility of unrelated compounds using CD rats from the same supplier, convulsions were observed in three rats, two of which were controls. Therefore, it is not known at this time whether the test material caused convulsions in the present study, contributed to the onset of convulsions in rats with a genetic predisposition for this condition, or whether the convulsions were a spurious finding due to a genetic anomaly in these animals and were unrelated to the test material.
Under the conditions of this study, the no-observed-effect level (NOEL) for males was 10 mg/kg/day. The NOEL for females was not determined.
Referenceopen allclose all
Mean Dose Consumed
- 1.5 % dose group: The mean feed consumed was 455 and 499 mg/kg bw/day for females 4825 and 4826, respectively.
- 3.0 % dose group: The mean feed consumed was 1785 and 1388 mg/kg bw/day for females 4827 and 4828, respectively.
Table 1: Body Weights (kg)
Concentration of the test material |
1.5 % |
3.0 % |
||
Animal No. |
4825 |
4826 |
4827 |
4828 |
Pre-test |
7.768 |
7.771 |
7.487 |
7.700 |
Day 2 |
7.777 |
7.819 |
7.324 |
7.746 |
Day 4 |
8.146 |
7.874 |
7.590 |
7.646 |
Day 8 |
8.279 |
7.980 |
8.040 |
8.044 |
Day 11 |
8.564 |
8.273 |
7.899 |
8.131 |
Day 15 |
8.534 |
8.258 |
8.228 |
8.204 |
Table 2: Food Consumption (g/day)
Concentration of the test material |
1.5 % |
3.0 % |
||
Animal No. |
4825 |
4826 |
4827 |
4828 |
Pre-test |
377 |
485 |
382 |
319 |
Day 2 |
213 |
276 |
298 |
328 |
Day 4 |
240 |
223 |
570 |
353 |
Day 8 |
274 |
290 |
SC |
SC |
Day 11 |
276 |
SC |
446 |
379 |
Day 15 |
253 |
274 |
541 |
410 |
SC = feed scratched from feeder
Table 1: Test Material Intake
Sex |
Week |
Dosage (mg/kg bw/day) |
||||
1 |
10 |
100 |
500 |
1000 |
||
Male |
1 |
1.089635 |
10.759157 |
110.827577 |
552.283566 |
1111.163780 |
2 |
1.026733 |
10.521698 |
99.412137 |
496.322096 |
1002.743396 |
|
3 |
0.995951 |
10.026385 |
93.878815 |
489.245902 |
973.514690 |
|
4* |
- |
- |
- |
- |
- |
|
Average |
1.037 |
10.436 |
101.373 |
512.617 |
1029.141 |
|
± SD |
0.0478 |
0.3739 |
8.6428 |
34.5338 |
72.5219 |
|
CV (%) |
4.61 |
3.58 |
8.53 |
6.74 |
7.05 |
|
% of target |
103.70 |
104.36 |
101.37 |
102.52 |
102.91 |
|
Female |
1 |
1.057564 |
10.733005 |
105.357980 |
533.632546 |
1050.729872 |
2 |
1.140165 |
11.173639 |
108.048193 |
522.941131 |
1109.261456 |
|
3 |
1.053616 |
10.803025 |
101.935388 |
505.086481 |
1006.698582 |
|
4* |
- |
- |
- |
- |
- |
|
Average |
10.84 |
10.903 |
105.114 |
520.553 |
1055.563 |
|
± SD |
0.0489 |
0.2368 |
3.0637 |
14.4220 |
51.4520 |
|
CV (%) |
4.51 |
2.17 |
2.91 |
2.77 |
4.87 |
|
% of target |
108.40 |
109.03 |
105.11 |
104.11 |
105.56 |
|
*A pre-terminal body weight was not collected on study animals. Therefore, the test material intake for week 4 of the study was not calculated.
Analytical Chemistry
To verify the mixing method and test material delivery, samples of the low- and high-dose diets were assayed for concentrations of the test material and homogeneity of mixing prior to the study start. Results indicated an acceptable agreement between actual and target levels. Percent relative standard deviations ranged from 2.57 to 3.19 % in the various samples tested, which confirmed the homogeneity of the mixing. The test material was shown to be stable in canine lab diet for at least 15 days at concentrations ranging from 0.3 to 3.0 %. Concentrations of the test material in the diets measured across the study averaged 90 to 96 % of target, which were considered to be acceptable results.
Table 1: Selected Haematologic Parameters
Dose (%) |
Animal No. |
Red blood cell count (x10^6/µL)
|
Haemoglobin concentration (g/dL) |
Haematocrit (%) |
0 |
5031 5032 |
7.34 7.40 (mean 7.37) |
15.7 16.3 (mean 16.0) |
47.5 50.0 (mean 48.8) |
Historical Controls |
- |
Range of 6.15 – 7.11 |
Range of 13.9 – 16.3 |
Range of 43.4 – 50.6 |
0.3 |
5033 5034 |
7.12 6.82 (mean 6.97) |
15.7 16.0 (mean 15.9) |
48.0 48.2 (mean 48.1) |
0.9 |
5035 5036 |
6.05 6.44 (mean 6.25) |
14.0 14.3 (mean 14.2) |
42.3 44.0 (mean 43.2) |
3.0 |
5037 5038 |
6.71 6.30 (mean 6.51) |
14.9 14.4 (mean 14.7) |
45.0 43.8 (mean 44.4) |
Analytical Chemistry
Samples of the low- and high-dose diets were assayed for concentrations of the test material and homogeneity of mixing prior to the study start, approximately mid-way through and near the end of the study to verify the mixing method and test material delivery. Percent relative standard deviations, which ranged from 2.07 to 8.74 % in the various samples tested, confirmed the homogeneity of the mixing, with the exception of one data point. The low-dose female diet analysed midway through the study had a relative standard deviation of 22.3 %. Considering that the low-dose female diet was mixed similarly throughout the study, and that homogeneity results at this dose level ranged from 5.03 to 8.74 % relative standard deviation for the other two homogeneity checks, this outlier did not have an adverse effect on the study.
Approximately 94.8 to 97.9 % of initial concentrations of the test material were present in diets after 27 days, indicating stability in the canine lab diet. Concentrations of the test material in the diets averaged 88.7 to 116 % of target indicating an acceptable agreement between actual and target levels.
Analytical Chemistry
To verify the mixing method, low- and high-dose samples collected from diets fed during week 1, and months 4, 9, and 12 of the study were assayed for homogeneity of test material in the diet. Percent relative standard deviations, which ranged from 1.82 to 10.8 % in the various samples tested (with a mean relative standard deviation of 4.2 %), confirmed the homogeneity of the mixing method. Samples of all dose levels prepared during week 1, and months 4, 9, and 12 of the study were assayed for concentration of the test material in the diet. The overall mean concentrations of the test material in the diets averaged between 97.8 and 101 % of target, indicating an acceptable agreement between actual and targeted levels.
Analytical Chemistry
The stability of the test material was established in a concurrent rat study. Diets containing the test material at 0.005 and 5 % in rodent feed were determined to be stable up to 42 days.
Homogeneity analyses were conducted on the high-dose male (1000 mg/kg bw/day) and the low-dose female diets (10 mg/kg bw/day) prior to study initiation. The results of the six random samples from each mixture indicated the preparations were homogeneously mixed based on relative standard deviations of < 10 %.
The concentration of the test material in all prepared diets including the premix and the control was determined prior to study initiation. Actual concentrations of the test material in the diets ranged from 88 to 112 % of targeted values and were judged to be within acceptable limits.
Overall Conclusion of Effects
There were no treatment-related effects in body weights, body weight gains, feed consumption, ophthalmologic, clinical, and gross pathological observations.
Haematological findings consisted of increases in platelet counts of males given ≥ 100 mg/kg bw/day, and were statistically identified. The differences between the control and the treated male groups may have reflected a treatment-related effect. However, these small differences were interpreted to be non-adverse. Cholesterol values were increased in both males and females treated with ≥ 100 mg/kg bw/day, statistically identified, and were outside of the historical control values. Although this finding was attributed to treatment, it was interpreted not to be an adverse effect. Absolute liver weights were increased in both males and females treated with ≥ 100 mg/kg bw/day and statistically identified. Relative liver weights were elevated in males treated with ≥ 100 mg/kg bw/day and females treated with ≥ 500 mg/kg bw/day. The increases in the absolute and relative liver weights in these treatment groups were attributed to the test material. Treatment-related histopathological changes in the liver consisted of hypertrophy and altered tinctorial properties of the hepatic lobule in males given 500 or 1000 mg/kg bw/day and one female given 1000 mg/kg bw/day. Males and females given 1000 mg/kg bw/day and males given 500 mg/kg bw/day also had slight hepatocellular vacuolation in the periportal region consistent with fatty change.
Table 1: Platelet Counts (Mean ± SD)
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
Platelets E3/UL |
1156 ± 102 |
1427 ± 111 |
1472* ± 130 |
1451* ± 82 |
1578* ± 133 |
1168 ± 226 |
1168 ± 210 |
1213* ± 176 |
1337* ± 100 |
1349* ± 153 |
Bold type indicates treatment-related effects.
* Statistical difference from control, males and females combined for analysis.
Table 2: Cholesterol Differences
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
Cholesterol mg/dl |
144 |
145 |
196* |
182* |
196* |
121 |
136 |
143* |
155* |
140* |
Bold type indicates treatment-related effects.
* Statistical difference from control, males and females combined for analysis.
Table 3: Organ Weight Differences
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
Liver (G) |
2.185 |
2.353 |
2.789* |
2.676* |
2.839* |
1.701 |
1.884 |
1.908* |
2.105* |
2.147* |
Liver (G/100) |
6.291 |
6.523 |
7.555* |
7.648* |
8.370* |
5.942 |
6.298 |
6.503 |
7.301* |
7.021* |
Bold type indicates treatment-related effects.
* Statistical difference from control, males and females combined for analysis.
Table 4: Treatment Related Microscopic Liver Effects
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
Hepatocellular hypertrophy with altered tinctorial properties, centrilobular/midzonal to panlobular |
1 |
0 |
1 |
4 |
4 |
0 |
0 |
0 |
0 |
1 |
Vacuolation, consistent with fatty change, periportal hepatocytes, very slight |
0 |
0 |
0 |
2 |
2 |
0 |
0 |
0 |
0 |
3 |
Bold type indicates treatment-related effects.
Analytical Chemistry
The homogeneity of the test material was evaluated in the low-dose female and the high- dose male diets from three time points during the pre-exposure and study interval. These data demonstrated coefficients of variation ranging from 4.25-7.93 %, indicating that the test material was homogeneously distributed within the feed.
The concentration of the test material in the diets from all dose levels, control and premix were evaluated three times during the pre-exposure and study interval.
Analyses indicated that the achieved overall concentrations of test material in all diets ranged from 93.3-105.7 % of targeted values, with individual means in the range of 91- 110 % of targeted values.
Table 1: Salient Treatment Related Effects
Sex |
Male |
Female |
||||||
Dose (mg/kg bw/day) |
3 |
30 |
300 |
1000 |
3 |
30 |
300 |
1000 |
↑ Mortality |
- |
1/10 |
1/10 |
4/10 |
- |
- |
3/10 |
5/10 |
↑ Liver Wt (g) |
- |
+ |
+ |
+ |
- |
+ |
++ |
++ |
↑ Liver Wt (g/100) |
+ |
+ |
++ |
++ |
- |
+ |
++ |
++ |
Hepatocyte hypertrophy |
+ |
++ |
+++ |
+++ |
- |
- |
+++ |
+++ |
Hepatocyte vacuolation |
- |
- |
+++ |
+++ |
- |
- |
+ |
+ |
Hepatocyte necrosis and/or inflammation |
- |
- |
+ |
+ |
- |
- |
- |
+ |
- effect not present, + to +++ reflect relative magnitude of the effect
Table 2: Clinical Chemistry, Treatment Related Effect
Sex |
Females |
||||
Dose (mg/kg bw/day) |
0 |
3 |
30 |
300 |
1000 |
Cholesterol mg/dL |
100 |
84 |
123 |
114 |
139* |
ALT U/L |
42 |
38 |
45 |
51 |
78 |
AST U/L |
107 |
90 |
119 |
90 |
133 |
Historical Control Data |
|||||
Cholesterol - 76 and 99 |
|||||
ALT - 41, 33, & 51 |
|||||
AST - 90, 85, & 115 |
|||||
Table 3: Organ Weights, Statistically Identified Differences
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
3 |
30 |
300 |
1000 |
0 |
3 |
30 |
300 |
1000 |
Final Body (g) |
39.8 |
39.8 |
39.0 |
38.0 |
38.4 |
29.6 |
31.0 |
31.0 |
32.7* |
32.6* |
Liver Wt (g) |
2.097 |
2.255 |
2.275 |
2.824$ |
2.815$ |
1.447 |
1.589 |
1.724* |
2.261* |
2.301* |
Liver Wt (g/100) |
5.281 |
5.672$ |
5.823$ |
7.393$ |
7.330$ |
4.879 |
5.135 |
5.552* |
6.907* |
7.030* |
% Increase Relative Liver Wt |
- |
7.41 |
10.3 |
40 |
38.8 |
- |
5.2 |
13.8 |
41.6 |
44.1 |
Bolded values interpreted to be treatment related. Statistically different from control mean by Wilcoxon's ($) or Dunnnett's (*) test, alpha = 0.05.
Table 4: Microscopic Treatment-Related Effects
Sex |
Male |
Female |
||||||||
Dose (mg/kg bw/day) |
0 |
3 |
30 |
300 |
1000 |
0 |
3 |
30 |
300 |
1000 |
Number of livers examined |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Hepatocyte hypertrophy, centrilobular/midzonal, diffuse, with altered tinctorial properties, very slight |
0 |
2 |
4 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Hepatocyte hypertrophy, centrilobular/midzonal, diffuse, with altered tinctorial properties, slight |
0 |
0 |
0 |
10 |
9 |
2 |
0 |
0 |
7 |
7 |
Hepatocyte vacuolation, consistent with fatty change, periportal, multifocal, very slight |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
4 |
2 |
Hepatocyte vacuolation, consistent with fatty change, periportal, multifocal, slight |
0 |
0 |
0 |
6 |
6 |
0 |
0 |
0 |
0 |
1 |
Necrosis with accompanying inflammation, focal or multifocal, very slight |
0 |
0 |
0 |
1 |
3 |
0 |
0 |
0 |
0 |
4 |
Inflammation, chronic, focal or multifocal, very slight |
0 |
0 |
0 |
4 |
3 |
0 |
0 |
0 |
0 |
0 |
Bolded values interpreted to be treatment related effects.
Analytical Chemistry
The homogeneity of the test material in rodent feed was determined for three separate mixing batches (mixed prior to study start, middle and near the conclusion of the study) for the 10 mg/kg bw/day female and 1000 mg/kg bw/day male test diets, the lowest and highest concentrations used in the study. The diets were homogeneous, with relative standard deviations for all diets sampled between 1.64 % and 6.37 %.
The concentrations of the test material were determined for the control, premix and test diets from all treatment levels at the three time points and were found to be acceptable. LC/MS analysis indicated 87 - 118 % of the target concentration of the test material for each individual sample. Target concentrations greater than 110 % were a single occurrence over the study for some diet mixes but mean values for the three analytical time points were within 98 – 108 %. The mean concentrations for each dose level ranged from 98.0 to 107.7 % of targeted concentration. No test material was found in the control diet.
The test material was stable for at least 42 days in the feed at concentrations ranging from 0.005 to 5 %.
Recovery Group Results
- Detailed clinical and Cage-Side Observations:
DCOs for recovery animals during the dosing phase of the study were addressed with the discussion for the main group animals (above). There were no DCOs conducted during the recovery phase of the study because there were no treatment-related effects in the main group animals. There were no treatment-related effects.
- Ophthalmology:
Ophthalmic findings for recovery animals observed during the dosing phase of the study were addressed with the discussion for the main group animals. There were no ophthalmic examinations conducted during the recovery phase of the study due to the absence of treatment-related effects in the main group animals.
- Body Weights and Body Weight Gains:
The lower body weights apparent at the end of the dosing period in the main group males (-3.9 %) and females (-3.3 %) given 1000 mg/kg bw/day were still present at the end of the 28-day recovery period in males (-4.6 %) and females (-4.4 %) given 1000 mg/kg bw/day during the dosing phase of the study.
- Feed Consumption:
Male rats given 1000 mg/kg bw/day consumed slightly less feed than the controls during the recovery phase of the study. These differences were not statistically identified and were interpreted not to be toxicologically significant. There were no significant differences in feed consumption for females.
- Prothrombin Time:
There were no significant differences in prothrombin time for male rats.
- Clinical Chemistry:
Female rats given 1000 mg/kg bw/day had a marginally higher ALP activity than the controls. This difference was statistically identified but was interpreted not to be treatment related because the value for the high-dose group was within the historical control data for recovery group female rats. The other chemistry values (ALP - males, AST, glucose, and total protein -males; cholesterol, calcium - females and albumin - females) were similar between control and high-dose rats indicating a reversal of effects seen following 90 days of exposure to the test material.
- Urinalysis:
Urine volume and specific gravity of males and females previously given 1000 mg/kg bw/day were similar to the controls. Male and female rats given 1000 mg/kg bw/day had a slightly higher pH than the controls. This may reflect the continued excretion of alkaline metabolites of the test material or interference with the test strip. There were no other differences in any of the urinary parameters for either males or females.
- Organ Weights:
The final body weights of males and females given 1000 mg/kg bw/day were lower than the controls and statistically identified. In addition, males given 1000 mg/kg bw/day had a higher relative brain weight, lower absolute epididymal weight, higher relative kidney weight, and higher relative liver weight; while females given 1000 mg/kg bw/day had higher absolute and relative adrenal weights, higher relative heart weight, higher absolute and relative liver weights, and lower absolute spleen weight.
The lower final body weights and increased liver and adrenal gland weights were interpreted to be treatment related and these effects did not completely return to control levels following 28 days of being given control feed. Final body weights for males and females given 1000 mg/kg bw/day were approximately 5 % lower and relative liver weights were 5 % and 13 % higher than the control values, while the relative adrenal weights for females given 1000 mg/kg bw/day were approximately 25 % higher than the controls. Lower body weight values were similar at 90 days and following 28 days of control feed. Increased relative liver weights decreased from ~20 % for males given 1000 mg/kg bw/day and 40 % for females given 1000 mg/kg bw/day at day 90 to values only 5 % and 13 % above controls at recovery day 28, indicating partial reversal of the liver weight increase. Adrenal weights (relative) decreased from ~33 % for females given 1000 mg/kg bw/day in the main group to 25 % above controls at recovery day 28, indicating a partial reversal of the adrenal weight increase.
The alterations in brain, kidney and heart weights were interpreted to be secondary to the lower body weight and were not target organs of the test material. A lower, absolute spleen weight in females was interpreted not to be treatment related since female rats given 1000 mg/kg bw/day, main group, from the dosing phase of the study had higher absolute spleen weights.
- Gross Pathology:
There were no treatment-related gross pathologic observations. All gross pathologic observations were considered spontaneous alterations, unassociated with exposure to the test material.
- Histopahology:
The livers of male and female rats given 0 or 1000 mg/kg bw/day from the recovery phase of the study were microscopically examined because treatment-related hepatocellular hypertrophy was noted in rats given 1000 mg/kg bw/day during the dosing phase of the study. Hepatocellular hypertrophy was noted in the livers of male rats but not female rats from the recovery phase of the study that were given 1000 mg/kg bw/day.
Treatment-related hypertrophy induced in females following 90 days of ingesting the test material was reversible after 28 days of control feed. A similar complete reversal did not occur in males given 1000 mg/kgbw/day, as hypertrophic changes were present in the majority of the males. Although not completely reversed, the effect was somewhat reduced in degree in that the initial hypertrophic changes involved the centrilobular to midzonal regions of the liver, whereas the effects following the exposure to control feed only involved the centrilobular region of the liver. The involvement of only the centrilobular region of the liver lobule versus the centrilobular and midzonal regions indicates some degree of reversibility and correlated with the lesser weight increase.
Conclusions
Table 1: Summary of Treatment Related Effects – Main Group
Dose (mg/kg bw/day) |
10 |
100 |
500 |
1000 |
↓ Body weight |
- |
MF |
MF |
MF |
↑ Liver weight (g) |
- |
MF |
MF |
MF |
↑ Liver, weight (g/100) |
MF |
MF |
MF |
MF |
↑ Adrenal weight (g) |
- |
F |
F |
F |
↑ Adrenal weight (g/100) |
- |
F |
F |
F |
↑ ALP |
- |
MF |
MF |
MF |
↓ AST |
- |
MF |
MF |
MF |
↓ Glucose |
- |
M |
M |
MF |
↑ Cholesterol |
- |
MF |
MF |
MF |
↑ Prothrombin time |
- |
M |
M |
M |
Microscopic liver hypertrophy with increased eosinophilia, centrilobular to midzonal |
M |
MF |
MF |
MF |
Electron microscopic increase in smooth endoplasmic reticulum |
ND |
ND |
ND |
MF |
↑ Liver mixed function oxidase enzymes |
ND |
ND |
ND |
MF |
- absence of an effect, M - male, F - female, ND = no data
Table 2: Summary of Salient Findings – Recovery Group
Recovery Status |
Complete Recovery |
Partial Recovery |
No recovery |
↓ Body weight (g) |
|
|
MF |
↑ Liver weight (g) |
M |
F |
|
↑ Liver weight (g/100) |
- |
MF |
|
↑ Adrenal weight (g) |
|
F |
|
↑ Adrenal weight (g/100) |
|
F |
|
ALP |
MF |
|
|
AST |
M |
|
|
Glucose |
MF |
|
|
Cholesterol |
MF |
|
|
Prothrombin time |
M |
|
|
Urine pH - alkaline |
|
F |
M |
Microscopic hepatocellula hypertrophy |
F |
M |
|
M - male, F - female
Analytical Chemistry
The homogeneity of the test material in rodent feed was determined on seven separate mixings (mixed prior to study start and at approximately 1.5, 3, 8, 12, 18, and 24 months) for the 0.1 mg/kg bw/day female and 300 mg/kg bw/day male diets, the lowest and highest concentrations used in the study. Homogeneity analyses were also conducted on the female 0.1 mg/kg bw/day diets (mixed on 1/14/03 and 5/31/03) and on the female 0.1 mg/kg bw/day and male 300 mg/kg bw/day diets (mixed on 12/01/03), to address homogeneity values from the previous analysis that were outside the laboratory’s acceptable range of ± 15 %. Follow-up analyses on diet mixes using the same procedures resulted in relative standard deviations (RSD) of < 10 %, indicating the diets were homogeneous, with the exception of the female 0.1 mg/kg bw/day diet that had a reported RSD of 18.2 % (analysed on 10/6/03). No specific reason for the variability in homogeneity of the 0.1 mg/kg bw/day female diets was determined. The overall RSDs for the female 0.1 mg/kg bw/day and male 300 mg/kg bw/day diets were 15.1 % and 4.08 %, respectively; therefore, the diets containing the test material were considered to be homogeneous.
The concentrations of the test material were determined for the control, premix, and diets from all treatment levels on seven separate mixings (mixed prior to study start and at approximately 1.5, 3, 8, 12, 18, and 24 months) and were acceptable. Analyses were also conducted (1/14/03, 5/31/03, and 12/01/03) to address homogeneity issues (previously described above) and were conducted (5/31/03 and 12/01/03) to address high-dose female and low-dose female concentration values outside the laboratory’s acceptable range of ± 15 %. Follow-up analyses on diets prepared from similar procedures resulted in acceptable target concentrations for these diets. Analytical results varied from 86.8 % to 123 % of the target concentration of the test material for individual samples, but only three analysed concentrations had a difference greater than 15 % from target. Mean concentrations for each dose level for all analytical time points ranged from 96.3 % to 105 % of target.
Stability of the test material was determined in the 0.1 mg/kg bw/day diet (0.0001 %, lowest concentration). The test material was stable in the 0.1 mg/kg bw/day diet for at least 27 days, at which time it was 111 % of the initial concentration.
Subchronic Toxicity
- Detailed clinical observations: There were no observations that were interpreted to be treatment related.
Clinical observations: There were no observations that were interpreted to be treatment related.
Ophthalmological examinations: There were no observations that were interpreted to be treatment related.
- Body weight and body weight gain: Body weight and body weight gains of rats given 1.0 mg/kg bw/day were comparable to controls.
- Feed consumption: Feed consumption for males (n = 10) given 1.0 mg/kg bw/day was higher and routinely statistically identified when compared to controls. This higher feed consumption was not observed when feed consumption for all animals on study was analysed at these time points (n = 75, Table 53) and was interpreted to be a result of the smaller sample size analysed in the subchronic study (n = 10/dose) and not a treatment-related effect. Feed consumption for females was comparable to controls.
- Test material intake: The test material intake was consistent with the targeted concentrations for the 1.0 mg/kg bw/day dose level over the course of the subchronic portion of the study. The actual dose received for males and females dosed at 1.0 mg/kg bw/day was 1.03 ± 0.08 and 1.01 ± 0.07 mg/kg bw/day.
- Haematologic (including prothrombin time): There were no statistically identified differences and no differences related to treatment.
- Clinical chemistry: There were no statistically identified differences and no differences related to treatment.
- Urinalysis: A higher urine volume and lower urine specific gravity occurred in females given 1.0 mg/kg bw/day. Similar alterations did not occur in females given 1.0 mg/kg bw/day at 3, 12, 18, and 24 months in the chronic toxicity/oncogenicity phase of the study. Therefore these differences were interpreted not to be treatment related.
- Organ and terminal body weights: There were no statistically identified differences in organ or final body weights for males given 1.0 mg/kg bw/day. The relative kidney weight and the absolute and relative ovary weights of females given 1.0 mg/kg bw/day were higher than the controls and statistically identified. Similar alterations in organ weights were not observed in females given 1.0 mg/kg bw/day for 12 months. The lack of repeatability within this study following a more prolonged administration of the test material indicated that the differences at 3 months were not treatment related.
- Gross pathology: The majority of males and females given 0 or 1.0 mg/kg bw/day had no visible lesions. Isolated rats had hiatal hernias and strangulated or necrotic mesenteric fat. These differences were attributed to spontaneous disease processes and were interpreted not to be treatment related.
- Histopathology: There were no treatment-related liver effects. Liver observations were the result of spontaneous alterations that commonly occur in this age and strain of rat. One gross lesion was suspected to be a portion of strangulated fat in a female given 1.0 mg/kg bw/day. This was determined to be a portion of strangulated/necrotic fat and was interpreted not to be treatment related.
Histopathology (non-neoplastic and neoplastic)
> 12 Months:
- Treatment Related Effects
Treatment related histological findings were confined to the lungs and liver. The livers of males given 300 or 75 mg/kg bw/day had slight centrilobular to midzonal hepatocyte hypertrophy and this change corresponded with the statistically identified increases in the liver weights.
In the lungs of males and females given 300 or 75 mg/kg bw/day, there were small areas of very slight or slight chronic inflammation either focal or multifocal, distributed in one or more lobes, which corresponded to the pale foci, observed grossly. Generally, these foci consisted of aggregates of large foamy alveolar macrophages variably admixed with small numbers of neutrophils and occasionally some lymphocytes.
Portions of the alveolar walls within these foci were variably thickened with increased amounts of collagen, infiltrating inflammatory cells with or without hyperplastic type-2 pneumocytes and macrophages. Additionally, in some animals, cholesterol clefts surrounded by macrophages were also seen in these foci of chronic inflammation. The grades very slight and slight were used to indicate the severity and/or extent of distribution (approximately 1 % or less, and 1-3 % of pulmonary parenchyma for grades ‘very slight’ and ‘slight’, respectively) but were clearly not adverse or detrimental to the overall health of the animals.
Two males given 300 mg/kg bw/day had benign hepatocellular adenomas in the liver. This was interpreted to be likely related to treatment due to 1) the absence of its occurrence in 12- months historical control data from three recent 2-year studies done in this laboratory and 2) elevated incidence of hepatic adenomas at 24 months (see Histopathology 24 Months section).
- Effects Not Related To Treatment
Eosinophilic foci (1-5 range) were seen in the livers of one male given 75 and in one male given 300 mg/kg bw/day. This low incidence and lack of dose response, and its absence in the females indicated that it was not treatment related. Similarly, there was a higher incidence of basophilic foci (6-10 range) in males given 75 mg/kg bw/day (4 of 10), but only one male given 300 mg/kg bw/day showed the same change. Again, this lack of dose response indicated that the higher incidence of basophilic foci (6-10 range) in males given 75 mg/kg bw/day was not related to treatment.
Very slight or moderate focal alveolar epithelial hyperplasia was seen in the lungs of one male given 75 mg/kg bw/day, one male given 300 mg/kg bw/day, and in one female given 300 mg/kg bw/day. Although this change was not seen in the controls, the low incidence in the 75 and 300 mg/kg bw/day dose groups was indicative that this alteration was not treatment related.
All other histopathologic observations, with the exception of the optic nerve lesions observed in rats from the 12- month interim necropsy were interpreted to be spontaneous changes typically seen in Fischer 344 rats of this age and husbandry conditions. The degeneration of the optic nerve was generally unilateral, and of varying severity including some in which little or no nerve tissue was even found among the fragments of connective tissue and retrobulbar vasculature (termed decreased size, severe). There was no relationship between the optic nerve lesions and exposure to the test material. The degeneration of the optic nerve was secondary to the repeated retro-orbital blood collection. In some of the rats, these iatrogenic optic nerve lesions were also accompanied by unilateral atrophy of the optic tract in the brain and unilateral retinal atrophy in the eye.
Neoplastic changes, both malignant and benign were observed at the 12- month interim necropsy, but were considered unrelated to exposure to the test material due to low incidence and/or lack of dose response. There were two instances of malignancy in the males of the 300 mg/kg bw/day dose group. They were 1) a poorly differentiated carcinoma in the mediastinal tissue, with metastasis to the lung and invasion into the adjacent skeletal musculature surrounding the thoracic vertebrae in one male and, 2) a thyroid follicular cell adenocarcinoma (without metastasis) in another male. Another instance of malignancy, which was an invasive rhabdomyosarcoma (without metastasis) of the diaphragm extending into the mesenteric and mediastinal tissue was observed in one control female (animal # 02A2147) that was humanely euthanised on day 232 due to its moribund status. Benign tumors diagnosed and interpreted to be unrelated to treatment, included a bronchiolo-alveolar adenoma in the lung of one female given 300 mg/kg bw/day, a cortical adenoma in a control female, ganglioneuroma of the adrenal gland in a female given 75 mg/kg bw/day, adenomas of the pituitary gland in 2, 1, and 2 males given 0, 1, or 75 mg/kg bw/day, respectively, and in two females given 0.1 mg/kg bw/day, and endometrial stromal polyps in the uteri of 3, 1, and 1 females given 0.1, 1, or 300 mg/kg/day, respectively.
> 24 Months:
- Treatment Related Effects (Neoplasms)
A higher incidence of hepatocellular adenomas was observed in the livers of males given 300 mg/kg bw/day (16 animals) relative to the controls (6 animals) and was statistically identified. The increase in the incidence was largely driven by higher incidence of males with one or two hepatocellular adenomas/animal. In addition, one male given 300 mg/kg bw/day had three hepatocellular adenomas. The liver tumor data indicated that the higher number of hepatocellular adenomas was not associated with an increase in the incidences of hepatocellular carcinomas, as the incidence of hepatocellular carcinomas between males given 0 or 300 mg/kg bw/day were comparable.
Historical control data from three recently completed 2-year studies in this laboratory show the range of the total number of males with hepatocellular adenomas was 1 or 2 out of 50 or 55 animals.
Since the incidence of hepatocellular adenomas in the 300 mg/kg bw/day dose group was higher than the concurrent control and historical control values, this change was interpreted to be treatment related. It should be noted that the incidence of hepatocellular tumors in the control group of this study was three times higher than that observed in the historical controls. The reason for this difference in background liver tumor incidence in control males and the significance of this apparent drift in the background liver tumor incidence was not determined.
Historical control liver tumor incidence from the National Toxicology Program (NTP) and Charles River Laboratories, indicates that the Toxicology & Environmental Research & Consulting historical control liver tumor data are similar to the mean data observed at other institutions.
Although hepatocellular carcinomas were seen in some animals, the incidence was low across all dose groups, namely 1, 1, 1, 0, and 2 males given 0, 0.1, 1.0, 75, or 300 mg/kg bw/day, respectively, and hence were interpreted to be unrelated to treatment.
Females given 300 or 75 mg/kg bw/day had a higher incidence of uterine polyps than the control females that were statistically identified. The increase in incidence was primarily due to a higher incidence of females having one and two uterine polyps/animal. The incidences of animals having three uterine polyps were somewhat similar with the controls having 1/50 and the 75 and 300 mg/kg bw/day groups having an incidence of 1/50 and 3/50, respectively.
In the historical controls from three recently reported two-year carcinogenicity studies, the range of the total number of females with endometrial stromal polyps was between 11 and 19, with some variability in the incidence of multiple polyps.
Historical control endometrial stromal polyp incidence data from the National Toxicology Program and Charles River Laboratories indicates that the Toxicology & Environmental Research and Consulting historical control data for endometrial stromal polyps are similar to the mean data observed at other institutions.
The incidences of the uterine polyps in females given 300 or 75 mg/kg bw/day were outside the concurrent and historical control data, suggesting that these differences were treatment related. Uterine stromal polyps were not observed with increased frequency in females given the test material at any dose level for 12 months, indicating that these uterine tumors did not have an early onset. At no time during the toxicologic evaluation of the test material was the uterus identified as a target organ in Fischer 344 rats (28 days – Lick et al., 1997; 90 days – Yano and Dryzga, 2002; and 12 months – see RESULTS previously discussed), Sprague-Dawley (two-generation reproduction study – Carney et al., 2004), CD-1 mice (28 days – Yano and Day, 2001, 90 days, Yano and Day, 2002 or 18 months - Johnson, 2005) or Beagle dogs (28 days – Stebbins et al., 2002, 90 days – Stebbins et al., 2003, one year – Stebbins et al., 2004).
Males and females given 75 or 300 mg/kg bw/day had lower incidences of leukemia than were observed in rats given 0, 0.1 or 1.0 mg/kg bw/day. The majority of these differences were statistically identified and were lower than historical control incidences. The mechanism for these differences was not determined.
- Treatment-Related Effects (Non-Neoplastic)
Treatment related histologic changes were observed in livers, lungs, and tail in both males and females, and in kidneys, testes and epididymides of males given the test material.
Treatment related hypertrophy of the hepatocytes was observed in males and females given 300 or 75 mg/kg bw/day and statistically identified. In the males, the hypertrophy was panlobular and in females it was mostly centrilobular/midzonal (with altered tinctorial properties – increased eosinophilia) in distribution. A few females given 300 or 75 mg/kg bw/day also had hepatocellular hypertrophy involving the entire hepatic lobule (panlobular); however, the difference in incidence between the controls did not clearly indicate that these differences were treatment related.
In the liver of males given 75 or 300 mg/kg bw/day, there was a treatment related increase in the incidence of basophilic foci (21 or more) relative to respective controls that was treatment related. The incidence of eosinophilic foci (6-10) was elevated in males given 75 mg/kg bw/day relative to that of controls, but due to the lack of dose response in the 300 mg/kg bw/day dose group, this was considered not treatment-related. In females, on the other hand, there was a decrease in the incidence of basophilic foci (21 or more) in the 75 and 300 mg/kg bw/day dose groups relative to the controls that were statistically identified. The relevance of these increases and decreases in foci was unclear.
As was noted at 12 months, the lungs of the majority of males and females given 75 or 300 mg/kg bw/day had small areas of very slight to slight chronic inflammation of alveoli, either focal or multifocal, distributed in one or more lobes, and generally corresponded to the pale foci observed grossly. These differences were statistically identified (multifocal slight, multifocal very slight and slight and all severities and distributions combined). In general, these foci consisted of aggregates of large foamy alveolar macrophages variably admixed with small numbers of neutrophils, occasionally some lymphocytes and multinucleated giant cells. Portions of the alveolar walls within these foci were variably thickened with increased amounts of collagen, infiltrating inflammatory cells with or without hyperplastic type-2 pneumocytes and macrophages. As a part of this chronic inflammation, small numbers of ciliated cells lined portions of alveolar walls in occasional animals. Additionally, cholesterol clefts surrounded by variable numbers of macrophages with or without occasional multinucleated giant cells were also seen, in some animals, within these foci of chronic inflammation. The grades very slight and slight were used to indicate the severity and/or extent of distribution (approximately 1 % or less, and 1-3 % of pulmonary parenchyma for grades ‘very slight’ and ‘slight’, respectively) but were clearly not adverse or detrimental to the overall health of the animals.
In the kidneys of males given 300 or 75 mg/kg bw/day, there were statistically identified higher incidences of unilateral hyperplasia of the pelvic epithelium and mineralisation of the pelvic epithelium. In general, these changes were mild, frequently focal and were interpreted to be treatment related.
Historical controls from three recent studies conducted in this laboratory indicated wide variations in the incidence of hyperplasia of the pelvic epithelium (1-11 out of 50-55) and mineralisation of the pelvic epithelium (1-19 out of 50-55). The combined incidence of very slight and slight hyperplasia of the pelvic epithelium in males given 300 mg/kg bw/day was elevated relative to the concurrent and historical controls and statistically identified, and this effect was considered treatment-related. However, although males given 75 mg/kg bw/day had statistically identified higher incidence of ‘very slight’ hyperplasia of the pelvic epithelium, it was within historical control range and the combined incidence across all grades was not statistically identified and hence, was considered not treatment-related.
The combined incidence of all grades of mineralisation of the pelvic epithelium of the kidneys were clearly elevated compared to controls and statistically identified in males given 300 or 75 mg/kg bw/day. In addition, its incidence was outside the historical controls and hence, these differences were considered treatment related.
In the testis, there was a statistically identified, increased incidence in severe bilateral atrophy of the seminiferous tubules in males given 300 mg/kg bw/day. This condition, in the majority of the affected animals was a secondary consequence to the presence of larger/heavier interstitial cell tumors (Leydig cell tumor) of the testis that corresponded to higher weights of testes in males given 300 or 75 mg/kg bw/day (absolute and/or relative testicular weights). Interstitial cell tumors are very common in Fischer 344 rats and nearly 100 % of Fischer 344 rats develop multiple hyperplasias or adenomas at the end of two-year studies (Boorman et al., 1990). Indeed, in the current study, the incidence of interstitial cell tumors (unilateral and bilateral combined), including controls, was 90 – 96 %.
Increased testicular weights (attributed to the interstitial cell tumors) and its secondary effect of increased incidence of severe bilateral atrophy of the seminiferous tubules were present in males given 300 mg/kg bw/day, and hence associated with treatment.
These effects likely reflect an exacerbation of a pre-existing high incidence of interstitial cell tumors and severe seminiferous tubule atrophy. Since effects on interstitial cells were not observed after 12 months of exposure to the test material, it appears that these alterations occur late in life. In addition, studies of variable duration evaluating the toxicity of the test material in Fischer 344 and Sprague-Dawley rats, CD-1 mice and Beagle dogs have not demonstrated any treatment-related effects involving the testes.
The consequence of the increased incidence of severe bilateral atrophy of the seminiferous tubules was evident in the epididymides in the form of an increased incidence of bilateral aspermia, findings which both occur commonly in aging Fischer 344 rats as evident in the concurrent control group. Statistically identified higher incidence of bilateral aspermia in the epididymides was seen in 37/49 and 38/49 males given 300 or 75 mg/kg bw/day. Again, this was interpreted generally to be a consequence of larger interstitial tumors (inferred from increased testicular weights), leading to an increased incidence of severe atrophy of seminiferous tubules with resultant lack of sperm in the epididymides of males given 300 or 75 mg/kg bw/day.
As mentioned earlier under the section ‘Gross Pathology – 24 Months’, there was an increased incidence of focal or multifocal lesions on the tails grossly identified as papules in males given 0.1, 75, and 300 mg/kg bw/day and females given 300 mg/kg bw/day (see Gross Pathology – 24 Month section). Histologically, the grossly recognised tail lesions were identified as focal or multifocal areas of hyperkeratosis with or without inflammation. In general, in the affected areas of the tail, there were variable degrees of focal/multifocal superficial dilated cystic structures lined by keratinised stratified squamous epithelium, and interpreted to be superficial hair follicles due to the presence of hair shafts and opening to the exterior. These hair follicles were generally distended with abundant keratin, some free hair shafts, variable amounts of inflammatory cells and debris, and frequently, these dilated follicles communicated with the surface of the skin. A chronic inflammatory response, when seen, was variably accompanied by focal epidermal hyperplasia, and focal dermal fibrosis. In some instances, focal epidermal ulceration and necrosis were also observed as part of the inflammatory process. The combined incidences of focal and multifocal hyperkeratosis in the tail of males and females given 300 mg/kg bw/day were interpreted to be treatment related, although the pathogenesis remains unclear.
Necrosis of the tail (tip) was another gross observation that was increased in incidence at necropsy mainly in males given 300 mg/kg bw/day. In general, in these affected tails, there was focally extensive chronic active inflammation, with or without ulceration. Usually the inflammation extended deep into the subcutis around a coccygeal vertebra, with reactive periosteal bone formation. In some cases there was inflammation of some arterioles causing thrombosis. In the affected area (tail tip) there was also full thickness necrosis of the tail tip involving the bone, bone marrow, connective tissue, and skin, consistent with infarction of the tail tip. Focally extensive inflammation in the tail of males given 300 mg/kg bw/day was statistically identified and considered related to treatment. In two males given 300 mg/kg bw/day, the histological diagnosis was ‘necrosis’ due to full thickness necrosis of the tail tip without any inflammation that was also considered a part of the spectrum of tail effects. The combined incidence of focally extensive inflammation and necrosis of tails in males given 300 mg/kg bw/day was increased and interpreted to be treatment related.
The thrombosis leading to infarction and necrosis of the tail tip was localised in nature, as there was no evidence of systemic thrombosis in other organs. The local thrombosis was consistent with a primary injury to the tail tip, leading to infection, moderate/severe focally extensive inflammation and necrosis.
As the tail was not a protocol required organ for routine histological analysis, only those tails that were grossly identified as having focal or multifocal papules or with other relevant gross observations at the time of 24- month necropsy were examined for histological correlation.
- Effects Not Related To Treatment (Non-Neoplastic)
In the kidneys, male rats given 1, 75, or 300 mg/kg bw/day had increased incidence of very slight and slight chronic progressive glomerulonephropathy (CPGN) that were statistically identified. CPGN is a common spontaneous geriatric kidney lesion in aging rats with males being much more affected than females (Montgomery and Seely, 1990; Short and Goldstein, 1992). Rat strains vary in the degree to which they spontaneously develop CPGN with Fischer 344 rats being one of the more affected strains.
The majority of the controls and those given 0.1 mg/kg bw/day had very slight CPGN (25 % or less involvement of the kidneys). 25/50, 25/50 and 28/50 of the male rats given 1, 75, or 300 mg/kg bw/day showed slight CPGN (26-50 % involvement of the kidneys), while the number diagnosed as moderate CPGN (51 -75 % involvement of the kidneys) was low and comparable across all dose levels including controls. Severe CPGN (> 75 % involvement) was seen in one male each in 75 and 300 mg/kg bw/day dose groups. The majority of females, on the other hand, showed only very slight CPGN across all dose groups including controls.
The apparent increase in the incidence of slight CPGN in males given 1, 75, or 300 mg/kg bw/day, although statistically identified, was considered not related to treatment for the following reasons:
1) Lack of convincing dose response. The fact that the incidence of slight CPGN remained virtually unchanged, with no progression to increased severity such as ‘moderate or severe’ with a 75 fold and 300 fold increase in dose levels.
2) The number of control males with ‘very slight’ CPGN in the present study (34) was higher as compared to many of the historical controls (0-33) and therefore, resulted in a proportional decrease in higher grades of severity as seen in the historical controls. Earlier studies done in this laboratory (except for one) within the past 10 years have shown variability in the incidence and severity of CPGN in control Fischer 344 rats at 24 months. Johnson et al., 2002, in his report on a 2-year carcinogenicity study, summarized historical controls on incidence and severity of CPGN in Fischer 344 rats observed in this laboratory (except for one).
Moreover, historical control data from three two-year studies conducted very recently in this laboratory under identical conditions indicated the wide variations in the incidence of severity. The incidence of slight CPGN in males given 1, 75, or 300 mg/kg bw/day in the current study (25–28) was in close proximity to the historical controls (11-25).
Females given 300 mg/kg bw/day had a higher incidence of severe unilateral atrophy of the optic nerve, unilateral cataract and severe unilateral atrophy of the retina, relative to control females and were statistically identified. Low incidences of severe unilateral atrophy of the optic nerve, unilateral cataracts, and severe unilateral retinal atrophy occurred in a non-dose related manner in females given 0.1, 1.0, and 75 mg/kg bw/day. The incidences of bilateral cataracts or bilateral severe retinal atrophy were low and did not indicate any treatment-related pattern. These data, in addition to the implausibility of a unilateral treatment-related effect on the optic nerve, lens, and retina strongly suggest that these differences were not treatment related and were likely the result of spontaneous disease processes commonly observed in Fischer 344 rats (Boorman et al., 1990).
Females given 75 or 300 mg/kg bw/day had higher incidences of very slight, multifocal atrophy of the pancreatic acini than the control females. The difference in incidence for females given 300 mg/kg bw/day were statistically identified. There was no increase in the incidence of acinar atrophy of slight, moderate, or severe degree indicating that the differences in the incidences of very slight, multifocal acinar atrophy were not treatment related and were likely the result of variability of spontaneous disease processes.
There were a number of other histopathologic lesions that were increased or decreased compared to the controls, and were statistically identified but were interpreted not to be treatment related for the following reason(s):
> Decreased Incidence of a Unilateral Lesion
Eye inflammation chronic active cornea unilateral focal – males given 300 mg/kg bw/day).
> Lack of a Dose-Response Relationship
Liver aggregates of macrophages/histiocytes, multifocal, very slight – males given 75 mg/kg bw/day
Eye mineralisation, cornea, unilateral multifocal, very slight – females given 75 mg/kg bw/day
Liver hypertrophy hepatocyte centrilobular/midzonal slight – females given 1.0 mg/kg bw/day
Liver necrosis hepatocyte multifocal – females given 0.1, 75, or 300 mg/kg bw/day
Liver vacuolisation consistent with fatty change individual cells – females given ≥ 0.1 mg/kg bw/day
Spleen extramedullary hematopoiesis – females given 300 mg/kg bw/day Uterus cyst endometrium multifocal – females given 75 mg/kg bw/day.
Table 2: Summary of Treatment Related Effects, 12Months– Males and Females
Dose (mg/kg bw/day) |
0.1 |
1.0 |
75 |
300 |
Body Weight / Weight Gain |
- |
- |
↓MF |
↓MF |
Liver Weight (G) & (G/100) |
- |
- |
↑MF |
↑MF |
Kidney Weight (G) & (G/100) |
- |
- |
↑M |
↑M |
Kidney Weight (G/100) |
- |
- |
↑F |
↑F |
Adrenal Weight (G) & (G/100) |
- |
- |
↑F |
↑F |
Heart G/100 |
- |
- |
↑M |
↑M |
Spleen G/100 |
- |
- |
- |
↑F |
Hepatocellular Hypertrophy |
- |
- |
↑MF |
↑MF |
Hepatocellular Adenomas |
- |
- |
- |
↑M |
Lung Inflammation |
- |
- |
↑MF |
↑MF |
M = males, F = females,↑ increased incidence or severity,↓ decreased incidence or severity. - = no effect.
Table 3: Summary ofTreatment RelatedEffects– Males and Females, 24 Months
Dose (mg/kg bw/day) |
0.1 |
1.0 |
75 |
300 |
Body Weight Gain - Males |
↑ 1.7 % |
↓ 2.5 % |
↓ 9.5 % |
↓ 13.9 % |
Body Weight Gain - Females |
↓ 2.1 % |
↑ 4.3 % |
↓ 21.8 % |
↓ 22.4 % |
Brain (G/100) |
- |
- |
↑ MF |
↑ MF |
Heart (G/100) |
- |
- |
↑ F |
↑ F |
Liver Weight (G) & (G/100) |
- |
- |
↑ MF |
↑ MF |
Kidney Weight (G) & (G/100) |
- |
- |
↑ M |
↑ M |
Adrenal Weight (G) & (G/100) |
- |
- |
↑ F |
↑ F |
Testes Weight (G) & (G/100) |
- |
- |
↑ M |
↑ M |
Spleen (G) & (G/100) |
- |
- |
↑ MF |
↑ MF |
Prothrombin Time |
- |
- |
↑ M |
↑ M |
Epididymides (G) & (G/100) |
- |
- |
↓ M |
↓ M |
Cholesterol |
- |
- |
↑ F |
↑ F |
Alkaline Phosphatase Activity |
- |
- |
↑ MF |
↑ MF |
Urine Volume |
- |
- |
↑ F |
↑ MF |
Urine Specific Gravity |
- |
- |
↓ MF |
↓ MF |
Hepatocellular Hypertrophy |
- |
- |
↑ MF |
↑ MF |
Hepatocellular Adenomas |
- |
- |
- |
↑ M |
Liver Foci, Basophilic, Hepatocyte (21 or more) |
- |
- |
↑M ↓F |
↑M ↓F |
Uterine Stromal Polyps |
- |
- |
↑ F |
↑ F |
Fischer Rat Leukemia |
- |
- |
↓ MF |
↓ MF |
Lung Inflammation |
- |
- |
↑ MF |
↑ MF |
Testes – Atrophy of Seminiferous Tubules |
- |
- |
- |
↑ M |
Epididymides – Aspermia |
- |
- |
↑ M |
↑ M |
Tail – Hyperkeratosis with or without Inflammation |
- |
- |
- |
↑ MF |
Tail Tip - Necrosis |
- |
- |
- |
↑ M |
Mineralization of Renal Pelvic Epithelium |
- |
- |
↑ M |
↑ M |
Hyperplasia of Renal Pelvic Epithelium |
- |
- |
- |
↑ M |
M = males, F = females,↑ increased incidence or severity, ↓ decreased incidence or severity. - = no effect.
Table 1: Group Terminal Body and Organ Weights
|
Dose group (mg/kg/day) |
|||||||||
Male |
Female |
|||||||||
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
|
Terminal Body weight (g) |
523.3 |
519.1 |
502.9 |
497.1 |
486.5 |
246.5 |
255.3 |
265.1 |
258.6 |
258.9 |
Liver (g) |
14.871 |
15.227 |
17.389 |
18.585* |
17.902* |
7.125 |
7.373 |
9.633* |
9.694* |
10.300* |
Liver (g/100) |
2.827 |
2.936 |
3.549* |
3.740* |
3.686* |
2.898 |
2.886 |
3.636* |
3.750* |
3.990* |
Adrenal (g) |
0.067 |
0.070 |
0.078 |
0.074 |
0.077 |
0.062 |
0.070 |
0.085* |
0.082* |
0.091* |
Adrenal (g/100) |
0.013 |
0.014 |
0.016* |
0.015 |
0.016* |
0.025 |
0.027 |
0.032* |
0.032* |
0.035* |
Epididymides (g) |
1.485 |
1.505 |
1.233* |
1.252* |
1.221* |
NA |
NA |
NA |
NA |
NA |
Epididymides (g/100) |
0.286 |
0.291 |
0.244* |
0.252* |
0.252* |
NA |
NA |
NA |
NA |
NA |
Values in bold indicate treatment-related effects
*Statistically different from control mean by Dunnett’s test, alpha = 0.05
Table 2: Incidence of Treatment-related Histopathologic Findings
|
Dose group (mg/kg/day) |
|||||||||
Male |
Female |
|||||||||
0 |
10 |
100 |
500 |
1000 |
0 |
10 |
100 |
500 |
1000 |
|
Liver: Hypertrophy; with altered tinctorial properties; centrilobular/midzonal; hepatocyte Very slight |
0 |
0 |
7 |
8 |
8 |
0 |
0 |
8 |
8 |
8 |
Liver: Vacuolisation; consistent with fatty change; periportal hepatocyte Very slight Slight |
0 0 |
0 0 |
1 0 |
1 0 |
1 0 |
0 0 |
2 0 |
3 2 |
3 2 |
3 3 |
Adrenal gland: Hypertrophy; zona fasciculata; diffuse Very slight Slight |
0 0 |
0 0 |
5 0 |
8 0 |
6 0 |
0 0 |
3 0 |
8 0 |
7 1 |
0 8 |
Epididymides: Decreased spermatic elements Very slight |
0 |
0 |
6 |
7 |
6 |
NA |
NA |
NA |
NA |
NA |
Epididymides: Degenerative spermatic elements Very slight Slight |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
NA |
NA |
NA |
NA |
NA |
Values in bold indicate effects considered to be related to treatment
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 1 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- The quality of the database is high. Subacute, subchronic and chronic studies performed to a high standard were available for assessment performed on multiple species.
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The subchronic and chronic toxicity of the test material was assessed in the key study Yano et al (2005).
Groups of 65 males and 65 female Fischer 344 rats were fed diets formulated to provide 0, 0.1, 1.0, 75, or 300 mg/kg bw/day for up to two years. An additional 10 rats/sex were given 0 or 1.0 mg/kg bw/day for 90 days to evaluate subchronic toxicity. Ten rats/sex/dose were necropsied after one year (chronic toxicity group), five rats/sex/dose were necropsied after one year (chronic neurotoxicity group) and the remaining 50 rats/sex/dose were fed the respective diets for up to two years and necropsied (oncogenicity group).
Daily cage-side examinations, weekly detailed clinical observations, and periodic body weight and feed consumption were monitored throughout the study on all rats.
Haematology, clinical chemistry and urinalysis parameters were evaluated at regular intervals throughout the study. Ophthalmic examinations were conducted on all rats pre-exposure and all surviving rats prior to the scheduled necropsies. All rats had a complete necropsy and organ weights were obtained from all rats at the scheduled necropsies. Histopathologic examinations of an extensive set of tissues were performed on all control and high-dose level rats and all rats that died spontaneously or were euthanized due to their moribund condition. Histopathologic examination of the surviving animals from the low- and intermediate-dose levels was limited to target organs, suspected target organs, and gross lesions.
Subchronic Toxicity: There were no treatment-related alterations observed in any parameter for male or females given 1.0 mg/kg bw/day, establishing this as a no-observed-effect level (NOEL) at 90 days.
Chronic Toxicity: Treatment-related effects consisted of: decreased body weight and body weight gains (males and females given 75 or 300 mg/kg bw/day), increased liver weights (males and females given 75 or 300 mg/kg bw/day), increased kidney weights (males given 75 or 300 mg/kg bw/day), and increased adrenal weights (females given 75 or 300 mg/kg bw/day). Treatment-related increases in the incidences of hepatocellular hypertrophy (males only) and lung inflammation were also observed in males and females given 75 or 300 mg/kg bw/day. Two males given 300 mg/kg bw/day also had hepatocellular adenomas that were treatment related. The NOEL was 1.0 mg/kg bw/day for both males and females at 12 months.
In the second key study, Yano and Dryzga (2002), a subchronic toxicity study, ten male and ten female Fischer 344 rats per group were given test diets formulated to supply 0, 10, 100, 500, or 1000 mg/kg bw/day for at least 90 days. Additional groups (ten/sex) were given either 0 or 1000 mg/kg bw/day for 90 days and were then given control feed for an additional 28 days to assess the reversibility of treatment-related effects induced following exposure to the test material. Parameters evaluated were daily observations, detailed clinical observations, ophthalmologic examinations, body weight, feed consumption, haematology, clinical chemistry, urinalysis, selected organ weights, gross and histopathologic examinations, and hepatic mixed function oxidase levels.
Male and female rats given 100, 500, or 1000 mg/kg bw/day had decreases in body weights (3-6 %) and body weight gains (4.7-13.4 %) compared to the controls which lacked a dose-response relationship. Liver weights of male and female rats given 10, 100, 500, or 1000 mg/kg bw/day were increased and statistically identified. Liver weight increases in rats given 100, 500, or 1000 mg/kg bw/day were not proportional to the dose administered. Microscopic effects consisting of an increase in hepatocytes size with an increase in eosinophilic staining of the hepatocyte cytoplasm occurred in males given ≥ 10 mg/kg bw/day and females given ≥ 100 mg/kg bw/day. Enzyme determinations indicated that the increases in liver weights were associated with a modest induction of a number of liver enzymes. In addition, the electron microscopic examination of the livers indicated that the smooth endoplasmic reticulum of centrilobular hepatocytes was increased in amount. Other effects of no or minor toxicologic significance occurred in male and/or female rats given ≥ 100 mg/kg bw/day during the dosing phase of the study and consisted of: 1) increased adrenal gland weights (females only), 2) minor increases in alkaline phosphatase activity, 3) minor decreases in aspartate aminotransferase activity and glucose levels (primarily males), and minor increases in cholesterol and prothrombin time (males only).
Complete recovery of treatment-related effects during the dosing phase of the study occurred with the following parameters: microscopic hepatocellular hypertrophy (females), and numerous alterations in clinical pathology parameters. A few of the parameters affected during the dosing phase of the study partially reversed during the recovery phase of the study and included: liver weights and adrenal weight increases, urine pH (females), and microscopic hepatocellular hypertrophy (males). The lower body weights identified during the dosing phase of the study persisted throughout the recovery phase of the study indicating that this effect was not reversible in 28 days.
Under the conditions of the test, the lowest-observed-effect level (LOEL) and no-observed-adverse effect level (NOAEL) were 10 mg/kg/day based on an increase in relative liver weights (males and females) and microscopic hepatocellular hypertrophy in males. A no- observed-effect level was not determined.
Two further studies in rats were available as supporting information, a subchronic study and a subacute (4 weeks) study. The supporting subchronic study, Zablotny et al (2002) was performed to a high standard to methods comparable to current guidelines and reported to a high level. The purpose of the study was to determine a suitable dosing regimen for a dietary reproduction study. The subacute study, Lick et al (1997) was performed to current guidelines, under GLP conditions. The gross pathological and histopathological changes observed at necropsy in both studies were consistent with the findings of the key studies. In the subchronic study, under the conditions of the test, the NOEL for males was 10 mg/kg/day, a NOEL for females could not be determined. In the subacute study the NOEL was the targeted concentration of 100 mg/kg bw/day and the NOAEL was the highest targeted concentration 1000 mg/kg bw/day for both males and females.
Further supporting studies were available in both mice and dogs. These are tabulated below:
Author (date) |
Study duration and Route |
Species and Strain |
Result |
Yano & Day (2001) |
28 day dietary study |
CD-1 mice |
NOAEL: 100 mg/kg bw/day (nominal) (males and females). Based on the lack of histopathological findings accompanying liver weight increases observed at this dose level. NOEL: 10 mg/kg bw/day (nominal) (males and females). Based on the increases in platelet count and cholesterol level in males at ≥ 100 mg/kg bw/day, and increases in liver weights in both males and females given ≥ 100 mg/kg bw/day. |
Yano & Day (2002) |
90 day dietary study |
CD-1 mice |
LOEL: 3 mg/kg bw/day (nominal) (males). Based on increased liver weights. LOEL: 30 mg/kg bw/day (nominal) (females). Based on increased liver weights. NOEL: 3 mg/kg bw/day (nominal) (females). Based on increased liver weights at 30 mg/kg bw/day (a NOEL could not be derived for the males). |
Baker (1997) |
14 days dietary study (palatability) |
Beagle dogs (females) |
Test material administered in the diet was found to be tolerable at 1.5 % or 3.0 %. No effect levels were determined. |
Stebbins et al (2002) |
28 day dietary study |
Beagle dogs |
NOEL: 3 % (males and females). No treatment-related effects observed in any of the study parameters examined up to the maximum dose level. 3.0 % was equivalent to 1034.9 and 1143.5 mg/kg bw/day in males and females, respectively. |
Stebbins et al (2002) |
90 day dietary study with 28 day recovery period. |
Beagle dogs |
NOEL: 0.003 % (males and females). Based on changes in organ weight (liver and kidney) and Haematological parameters (decreases in red blood cell count, haematocrit and haemoglobin concentration and increased mean corpuscular volume). Dose level was approximately equivalent to 1 mg/kg bw/day. |
Stebbins et al (2004) |
1 year dietary study |
Beagle dog |
NOEL: 0.003 % (males). Equivalent to 0.74 mg/kg bw/day. Based on increased mean corpuscular volumes and reticulocyte counts with polychromasia of red blood cells, erythroid cell hyperplasia of the bone marrow and increased platelet counts. Increased absolute liver weights in high dose males and higher relative liver weights at ≥ 0.03 %. Males receiving ≥ 0.03 % had increased lymphoid hyperplasia in the fundus and/or pylorus of the stomach. NOEL 0.03 % (females). Equivalent to 8.7 mg/kg bw/day.Increased reticulocyte counts with polychromasia of red blood cells in high-dose males and females. One female at 0.225 % had erythroid cell hyperplasia of the bone marrow. Increased platelet counts in high-dose females. Increased alkaline phosphatase activity was recorded in high dose females. Females dosed with 0.225 %, had increased mucosal lymphoid hyperplasia in the fundus and/or pylorus of the stomach. |
Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Repeated dose toxicity of the test material was evaluated using the chronic and subchronic toxicity studies available which were performed on the preferred species (rat). Studies were performed under GLP conditions and in line with current standardised guidelines. Both studies were performed and reported to a high standard and were therefore assigned a reliability score of 1, in line with the principles for assessing data quality, as described by Klimisch et al (1997). The studies were in agreement, and furthermore were supported by several further good quality studies. The chronic study was selected as a key study as it was the longest duration and therefore the preferred method, the subchronic study was selected on the basis that a NOAEL could be derived from the observed effects.
Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I (Classification and Labelling Requirements for Hazardous Substances and Mixtures), Regulation (EC) No. 1272/2008 (CLP), the substance does not require classification for specific target organ toxicity after repeated exposure.
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