Registration Dossier

Administrative data

Description of key information

90-Day rat feeding study LOAEL: 1250 ppm (equivalent to 104.9 and 120.1 mg/kg bw/day for males and females, respectively); OECD 408; Reliability = 1

90-Day mouse feeding study LOAEL: 800 ppm (equivalent to 137.3 and 176.1 mg/kg bw/day for males and females, respectively); OECD 408; Reliability = 1

90-Day dog feeding study LOAEL: 500 ppm males (equivalent to 8.9 and 16.9 mg/kg/day for males and females, respectively); OECD 409; Reliability = 1

1-Year dog feeding study LOAEL: 500 ppm (equivalent to 16.1 and 15.7 mg/kg bw/day for males and females, respectively); OECD 452; Reliability = 1

105-Week rat feeding study LOAEL: 3500 ppm (equivalent to 162.1 and 203.3 mg/kg bw/day for males and females, respectively); OECD 453; Reliability = 1

18-Month mouse feeding study LOAEL: 2400 ppm for males (equivalent to 293 mg/kg/day) and >4800 ppm for females (highest dose tested; equivalent to 799 mg/kg bw/day); OECD 451; Reliability = 1

28-Day rat inhalation study LOAEL: >0.025 mg/L (highest concentration tested); OECD 412; Reliability = 1

28-Day rat dermal study LOAEL: >1000 mg/kg/day (highest dose tested); OECD 410; Reliability = 1

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Qualifier:
according to guideline
Guideline:
EU Method B.30 (Chronic Toxicity Studies)
Qualifier:
according to guideline
Guideline:
EPA OPP 83-1 (Chronic Toxicity)
Qualifier:
according to guideline
Guideline:
other: Japanese Agricultural Chemical Laws and Regulations Japan (ii) (1985) Society of Agricultural Chemical Industry
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 94.4% (w/w)
Species:
dog
Strain:
Beagle
Details on species / strain selection:
The dog is the preferred non-rodent species for regulatory studies and the Alderley Park beagle was used because of the substantial background data available for this strain, in the Laboratory, relating to studies of this type.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Convential Animal Breeding Unit, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 16-24 weeks old on delivery
- Weight at study initiation: not provided
- Fasting period before study: no
- Housing: On arrival, dogs were housed by treatment group (sexes separately) in indoor pens. The pens have a sleeping platform with heated floor underneath and interlinking gates which enable the dogs to be separated for feeding. Dogs received exercise (while on a lead) for all routine procedures (weighing) and were allowed weekly access to a free exercise area, on a group and sex basis.
- Diet: 350 g for males and 300 g for females of the appropriate experimental diet/day
- Water: ad libitum
- Acclimation period: 4-5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19±4°C
- Humidity: 40-70%
- Air changes (per hr): Approximately 15 changes/hour
- Photoperiod (hrs dark / hrs light): Artificial giving 12 hours light, 12 hours dark
Route of administration:
oral: feed
Details on route of administration:
The dietary route was chosen for administration of the test substance as this represents a possible route of exposure in humans and other mammalian species.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared in 60 kg batches
- Mixing appropriate amounts: A 1 kg premix was prepared by addition of the test substance to a small amount of LABORATORY DIET A, whilst mixing in a automatic pestle and mortar, and then adding the remaining diet to make up the 1 kg premix. This was then mixed with 59 kg of diet in a blender. Water was added to the batch of diet at a rate of approximately 100 mL/kg diet and the diets mixed twice for 6 minutes in a Hobart mixer. The diet was then pelleted, using a Laboratory Pellet Mill Feeder.
- Storage temperature of food: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted with acetonitrile
Method: HPLC
HPLC Conditions
- Pump: 600 Series (Waters)
- Mobile phase: Acetonitrile (65% v/v); Water (35% v/v); Phosphoric acid (0.1% v/v)
- Flow rate: 1.5 mL/min
- Detector wavelength: 245 nm
- Column: 25cm x 4.6mm ID Hypersil BDS (Shandon) or 15cm x 3.9mm ID Symmetry C18 (Waters)
- Column temperature: Ambient
- Sample introduction: 717 plus (Waters)
- Injection volume: 20 µL
- Limit of detection: 0.2 µg/mL (corresponding to 2 ppm)
Mean measured concentrations achieved: 47.0, 140, and 485 ppm for 50, 150 and 500 ppm groups respectively. The overall mean concentrations were within 7% of target.
Homogenity and chemical stability were determined to be satisfactory.
Duration of treatment / exposure:
1 year
Frequency of treatment:
Daily
Dose / conc.:
50 ppm
Dose / conc.:
150 ppm
Dose / conc.:
500 ppm
No. of animals per sex per dose:
4/gender/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on the results of a previous study in the dog carried out in the test facility.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: Food residues were recorded daily, approximately 4 hours after feeding and any residual food was discarded. These measurements were made for at least 2 weeks pre-study and throughout the treatment period. Food consumption was calculated, at weekly intervals, as a mean value for each dog.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks -1, 13, 26, and prior to termination
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white cell count (WBC), differential WBC count, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks -1, 13, 26, and prior to termination
- Parameters examined: alanine aminotransferase activity, albumin, alkaline phosphatase activity, aspartate aminotransferase activity, calcium, chloride, creatinine, creatine kinase activity, gamma-glutamyl transferase activity, glucose, phosphorus (as phosphate), potassium, sodium, total bilirubin, cholesterol, total protein, urea

URINALYSIS: Yes
- Time schedule for collection of urine: weeks -1, 13, 26, and prior to termination
- Parameters examined: volume, appearance, colour (only if abnormal), specific gravity, pH, glucose, ketones, bilirubin, protein, blood. In addition, each urine sample was centrifuged and the sediment stained and examined microscopically to identify components.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to a full examination post mortem, which involved an external observation and a careful examination of all internal organs and structure. The following organs were removed, trimmed free of extraneous tissue and weighed: adrenal glands, brain, kidneys, liver (without gall bladder), ovaries, testes, thyroid glands with parathyroid.

HISTOPATHOLOGY: Yes (see table 1)
Statistics:
All data were evaluated using analysis of variance and/or analysis of covariance for each specified parameter using the GLM procedure in SAS.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was an increased incidence of thin appearance for females receiving 500 ppm. The only other clinical sign that was considered possibly to be related to treatment was an increased incidence of reddened gums for males receiving 500 ppm. There was an increased incidence of reddened ears in males receiving 500 ppm. Reddened ears were observed at all dose levels including controls and are considered to be incidental to administration of the test substance. There were no clinical signs associated with administration of 50 or 150 ppm.

The only gastrointestinal finding that was considered to be related to treatment was a slightly increased incidence of fluid faeces in males receiving 500 ppm.

None of the clinical signs related to treatment were considered to be of toxicoligical significance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related reduction in body weight was seen for males and females receiving 500 ppm. There were no effects on body weights at 50 or 150 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment-related effects on food consumption was seen for males and females receiving 500 ppm. There were no treatment-related effects on food consumption at 50 or 150 ppm.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no veterinary or ophthalmoscopy findings that were considered related to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Minor variations in haematology parameters were seen at isolated time points but these were small in magnitude and/or showed no dose response relationship and were considered to be incidental to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Minor variations in blood chemistry parameters were seen at isolated time points but these were small in magnitude and/or showed no dose response relationship and were considered to be incidental to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Minor variations in urine clinical chemistry parameters were seen at isolated time points but these were small in magnitude and/or showed no dose response relationship and were considered to be incidental to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean thyroid weights, adjusted for terminal body weight, for females receiving 500 ppm were higher than concurrent controls. The control group mean value was low compared to historical control group mean range. Whilst a relationship to treatment cannot be entirely ruled out, in the absence of any associated treatment-related histopathological changes the increase is considered to be of no toxicological significance.

Group mean kidney weights for males receiving 500 ppm were lower than concurrent controls but the reduction was not statistically significant after adjustment for terminal body weight and is considered to be incidental to treatment with the test substance. Group mean brain weights, for females receiving 500 ppm were slightly higher than concurrent controls but the increase did not attain statistical significance and is considered to be incidental to administration of the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slight dilatation of the lateral ventricles of the brain was observed macroscopically in one animal of each sex receiving 500 ppm and in one male receiving 150 ppm but on histopathological examination this was considered to be within normal limits. The small number of macroscopic changes observed in other tissues were considered to be incidental to administration of the test substance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A number of minor changes were observed but all were considered to be incidental to administration of the test substance.
Key result
Dose descriptor:
NOAEL
Effect level:
150 ppm
Sex:
male/female
Remarks on result:
other: clinical observations, body weight reduction, food consumption effects at 500 ppm
Key result
Critical effects observed:
no

Dose rates were calculated in terms of mg/kg/day. Mean values for males were: 1.6, 4.8, and 16.1 mg/kg/day for the 50, 150, and 500 ppm groups respectively. Mean values for females were 1.6, 4.6, and 15.7 mg/kg/day for the 50, 150, and 500 ppm groups respectively

Conclusions:
Administration of 500 ppm in the diet for 52 weeks resulted in reduced growth and reduced food consumption in males and females. This dose was equivalent to an overall mean dose of 16.1 mg/kg/day for males and 15.7 mg/kg/day for females. There were no other findings considered to be of toxicological importance. A dietary level of 150 ppm (4.8 mg/kg/day in males; 4.6 mg/kg/day in females) was without toxicologically significant effects in male and female dogs.
Executive summary:

Groups of 4 male and 4 female beagle dogs were fed diets containing 0, 50, 150, or 500 ppm for a period of at least 1 year according to OECD guideline 452. Clinical observations and veterinary examination (including ophthalmoscopy) were made and body weights, food consumption and clinical pathology parameters were measured periodically throughout the study. At the end of the scheduled period, the animals were killed and subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. Dietary administration of 500 ppm to male and female dogs resulted in reduced growth and reduced food consumption. Administration of 500 ppm to female dogs resulted in an increased incidence of thin appearance. There was an increased incidence of reddened gums and of fluid faeces in males receiving 500 ppm but these were considered to be of no toxicological significance. At necropsy, group mean thyroid weights, adjusted for terminal body weight, for females receiving 500 ppm were higher than concurrent controls, however, in the absence of any associated treatment-related histopathological changes the increase in thyroid weight is considered to be of no toxicological significance. There were no changes in clinical pathology parameters and no pathology findings attributed to treatment with the test substance. 

Administration of 500 ppm in the diet for 52 weeks resulted in reduced growth and reduced food consumption in males and females. This dose was equivalent to an overall mean dose of 16.1 mg/kg/day for males and 15.7 mg/kg/day for females. There were no other findings considered to be of toxicological importance. A dietary level of 150 ppm (4.8 mg/kg/day in males; 4.6 mg/kg/day in females) was without toxicologically significant effects in male and female dogs.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
99% w/w
Species:
mouse
Strain:
other: C57BL/10JfAP/Alpk
Details on species / strain selection:
This strain of mouse was used because of the substantial background data available for this strain, in the test facility, relating to studies of this type.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Zeneca Pharmaceuticals, Alderley Park
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:weanlings (29-31 days old on delivery)
- Weight at study initiation: not provided
- Fasting period before study: no
- Housing: Mice were housed, sexes separately, in multiple mouse racks suitable for animals of this strain and weight range expected during the course of the study. They were housed up to 9 per cage, sexes separately, initially and in fives after they had been assigned to experimental groups.
- Diet: ad libitum
- Water:ad libitum
- Acclimation period: miniumum of 5 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of CT1 diet is routinely analysed for composition and for the presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 40-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light): Artificial giving 12 hours light, 12 hours dark
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from all dietary levels (including controls) were taken at intervals throughout the study and analysed quantitatively for the test substance. The homogeneity of the test substance in CT1 diet was determined by analysing samples from the low and high dose levels. The chemical stability of the test substance in diet was determined at these same dose levels at room temperature over a period of up to 78 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 ppm
Dose / conc.:
200 ppm
Dose / conc.:
800 ppm
Dose / conc.:
1 600 ppm
Dose / conc.:
2 400 ppm
No. of animals per sex per dose:
10/gender/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels selected were based on the results of a 28-day feeding study in the mouse carried out in the test facility.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 1-8 inclusive and then on the same day, were practicable, of each subsequent week until termination.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1-8 inclusive and then on the same day, were practicable, of each subsequent week until termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes Days 1-7 for each cage of mice and calculated as a weekly mean for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1) From all animals surviving to scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed: adrenals, brain, kidneys, liver, testes

HISTOPATHOLOGY: Yes (see table 1)
Statistics:
All data were evaluated using analysis of variance and covariance for each specified parameter using the GLM procedure in SAS. Analysis of variance was used, separately for males and females, for food consumption and food utilization parameters. Analysis of covariance was used, separately for males and females, for body weight data and organ weight data.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical effects considered to be related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males given 2400 or 1600 ppm showed a reduction in final body weight, compared to control, of approximately 6-7%. Females given 2400 ppm showed a reduction in final body weight, compared to control, of approximately 10% and females given 1600 or 800 ppm showed a reduction in final body weight compared to control, of approximately 4-6%. Animals of both sexes given 200 ppm and males at 800 ppm had body weight values similar to controls throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced initially in both sexes given 2400, 1600 or 800 ppm but had converged to control values by the end of the first week of the study. Thereafter food consumption remained similar to controls throughout the study. Female food consumption values were more variable throughout the study than male values fan for all treated groups were generally slightly lower than control from week 9 of the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food utilization was less efficient in both sexes given 2400 ppm during days 1-28 and overall and in males given 1600 ppm during days 1-28 and overall. Females given 1600 ppm showed less efficient food utilization during days 1-28 but this was due to an effect in one cage of animals and is probably of no toxicological significance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Both sexes given 2400, 1600 or 800 ppm showed increases in liver weight, after adjustment for terminal body weight, compared to control animals. All other organ weights in treated groups were similar to control organ values.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The only apparent treatment related change was the presence of an increased incidence over expected background of dark areas/spots in the spleens of male mice dosed at 2400 ppm. Other changes were either not treatment related or consistent with changes expected for this age and strain of mouse.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related significant increase in minimal hepatocyte hypertrophy in the livers of males given 1600 or 2400 ppm and of females given 800, 1600 or 2400 ppm. 2/10 males at 800 ppm and 2/10 females at 200 ppm were also affected but this was not significant. The change observed was subtle, not clearly increased in severity by increasing dose and of doubtful toxicological significance.
In addition, there was an increase in focal pigmentation of the spleens in 4/10 male mice given 2400 ppm. Perl’s stain showed this to be negative for haemosiderin and was therefore interpreted as being melanin. Since this strain of mouse is a heavily pigmented strain and since focal melanin deposits in the spleen are not an uncommon occurrence in this strain, this change is considered to be of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
200 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
Oral administration of 2400 ppm for 90 consecutive days adversely affected food consumption and body weight gains and resulted in increases in liver weight and changes in liver pathology. Some or all of these changes were seen at dose levels of 160 or 800 ppm but the severity and incidence of these changes were generally reduced. The no-observed adverse effect level for dietary administration was considered to be 200 ppm.
Executive summary:

Groups of 10 male and 10 female mice were fed diets containing 0, 200, 800, 1600, or 2400 ppm for 90 consecutive days. Clinical observations, body weights and food consumption were measured and at the end of the scheduled period, the animals were killed and subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. There were no clinical changes considered related to the test substance. There were reductions in food consumption and body weight, compared to concurrent control animals, and increases in liver weight, after adjustment for terminal body weight, in animals at dose levels of 2400, 1600, or 800 ppm. Food utilization was less efficient in animals given 2400 or 1600 ppm. There was a treatment related increase in incidence of minimal hepatocyte hypertrophy in the livers of males given 2400 or 1600 ppm and in livers of females given 2400, 1600, or 800 ppm. The no-observed adverse effect level for dietary administration was considered to be 200 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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)
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 93.3% (w/w)
Species:
rat
Strain:
other: Alpk:APfSD
Details on species / strain selection:
This strain of rat was used because of substantial background data available for this strain, in the test facility, relating to studies of this type.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Zeneca Pharmaceuticals, Alderley Park
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Weanlings (approximately 22 days old on delivery)
- Fasting period before study: Not provided
- Housing: The rats were housed, sexes separately, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of the study. They were housed in litters initially and in fours after they had been assigned to experimental groups.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Each batch of CT1 diet was routinely analyzed for composition and for the presence of contaminants. Water was also periodically analyzed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 40-70%
- Air changes (per hr): At least 15 changes/hour, giving positive pressure with respect to the access corridor
- Photoperiod (hrs dark / hrs light):Artificial giving 12 hours light, 12 hours dark
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Not provided
- Mixing appropriate amounts with (Type of food): The experimental diets were made in 30 kg batches from premixes by triturating the appropriate amount of test substance with 500 g of diet. The premixes were then added to 29.5 kg of diet and mixed thoroughly. All diets were based on CT1 diet supplied by Special Diet Services Limited, Stepfield Witham, Essex, UK.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from all dietary levels (including controls) were taken at intervals throughout the study and analyzed quantitatively. The homogeneity of test substance in the diet was determined by analyzing samples from the low and high dose levels. The chemical stability of the test substance was determined at these same dose levels over a period of up to 89 days.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Dose / conc.:
100 ppm
Dose / conc.:
500 ppm
Dose / conc.:
1 250 ppm
No. of animals per sex per dose:
12/gender/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected for this study based on results of a preliminary dose range finding study in the Alpk:APfSD rat carried out in the test facility.
- Rationale for animal assignment: Random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Immediately before feeding of the experimental diets commenced and then on the same day, where practicable, of each subsequent week until termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
- Time schedule for examinations: Continuously throughout the study for each cage of rats and calculated as weekly mean for each cage.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Eyes of all animals were examined pre-experimentally and groups 1 and 4 (0 and 1250 ppm) were examined during the week prior to termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: All surviving animals
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, red cell distribution width, total white cell count (WBC), differential WBC count, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:At study termination
- Animals fasted: Not specified
- How many animals:All surviving animals
- Parameters examined: alanine aminotransferase, alkaline phosphatase , aspartate aminotransferase activity, calcium, chloride, creatinine, creatine kinase , gamma-glutamyl transferase , glucose, phosphorus (as phosphate), potassium, sodium, total bilirubin, cholesterol, total protein, urea, triglycerides, albumin

URINALYSIS: Yes
- Time schedule for collection of urine: the week prior to termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, appearance, colour, specific gravity, pH, glucose, ketones, bilirubin, protein, blood. In addition, each urine sample was centrifuged and the sediment stained and examined microscopically to identify components.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to a full examination post mortem. This involved an external observation and a careful internal examination of all organs and structures. The following organs were removed, trimmed free of extraneous tissue and weighed: adrenal glands, brain, epididymides, kidneys, liver, testes

HISTOPATHOLOGY: Yes (see table 1)
Statistics:
All data were evaluated using analysis of variance and/or analysis of covariance for each specified parameter using the GLM procedure in SAS.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in any treated animals that were considered to be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male given 1250 ppm was terminated for humane reasons, unrelated to treatment, in week 7 of the study. There were no other mortalities.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats given 1250 ppm showed an initial decrease in body weight, when compared to control values, of approximately 7% and 4% in males and females respectively during the first week of the study; this deficit reached 10% and 8% in males and females respectively by the end of the study. Body weights in all other treatment groups were within 5% of control values throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in males and females given 1250 ppm was decreased, when compared to controls throughout the study. Food consumption in both sexes given 100 or 500 ppm was generally similar to control values throughout the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food utilization was less efficient in females given 1250 ppm during the first four weeks of the study but thereafter was similar to control values. Food utilization efficiency in males at this dose level and in both sexes given 100 or 500 ppm was similar to controls throughout the study.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no changes that were considered to be related to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males given 1250 or 500 ppm showed a slight reduction in mean cell volume and in both sexes at these dose levels there was a reduction in mean cell haemoglobin. Females given 1250 ppm showed a very slight reduction in mean cell haemoglobin concentration. There were increases in white blood cell (neutrophil, lymphocyte, and monocyte) counts in females given 1250 ppm but this was largely due to increases in one animal. The deviations from the control group values seen in this animal are considered not to be treatment related and therefore this animal was excluded from the statistical analysis; the values of the remaining animals in the group were similar to controls.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were reductions in plasma alkaline phosphatase, alanine and aspartate aminotransferase activities and plasma triglycerides in males given 1250 ppm and in plasma alkaline phosphatase activities in females at this dose level. Plasma alkaline phosphatase activities were reduced to a lesser extent in males given 500 ppm. Females given 1250 ppm showed a slight increase in plasma bilirubin and plasma cholesterol; the latter change was also present in females given 500 ppm. Reduction in some enzyme activities in males and feamels were minor and not considered to be of toxicological importance.

The plasma urea levels were slightly raised in males given 500 ppm and in both sexes given 1250 ppm. In the absence of any change in plasma creatinine levels, this finding is considered to be of little toxicological importance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects or trends seen on either urinalysis or urine ctology profiles.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was no increase in absolute liver weight. However, there was an increase in lever weight, after adjustment for terminal body weight, in both sexes given 1250 ppm and in males given 500 ppm. In absence of any change in the histopathology of the liver, this effect is not considered to be of toxicological significance.

Other organ weights in all treated groups were generally similar to control values. There were some deviations from control values but these were slight and/or showed no evidence of a coherent dose-response and are considered to be unrelated to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The 1250 ppm male terminated during week 7 of the study showed distension of the intestines. Macroscopic changes seen in all other animals are considered to be typical in this strain of rat and unrelated to treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The 1250 ppm male terminated during week 7 of the study showed a marked granulomatous inflammation of the colon, with spread of a necrotic process to one lobe of the liver, the changes seen in the liver being consequential to those of the colon. These changes were considered not to be due to treatment. Microscopic changes in all other animals are considered not to be related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
Key result
Critical effects observed:
no

Dose rates were calculated in terms of mg/kg/day. Mean values for males were: 8.5, 41.7, and 104.9 mg/kg/day for the 100, 500, and 1250 ppm groups respectively. Mean values for females were 9.7, 48.1, and 120.1 mg/kg/day for the 100, 500, and 1250 ppm groups respectively.

Conclusions:
Oral administration of 1250 ppm for 90 consecutive days produced signs of toxicity as evidenced by decreases in body weight and food consumption/utilization efficiency. Oral administration of 500 ppm produced no effects that were considered toxicologically significant.
Executive summary:

Groups of 12 male and 12 female rats were fed diets containing 0, 100, 500, or 1250 ppm for 90 consecutive days according to OECD guideline 408. Clinical observations, body weights and food consumption were measured and, at the end of the scheduled period, the animals were killed and subjected to a full examination post mortem. Cardiac blood samples were taken for clinical pathology. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. There were no treatment-related mortalities. In animals surviving to scheduled termination there were no treatment-related changes in clinical condition or ophthalmoscopy and no treatment-related histopathological changes in any treated group. Animals of both sexes given 1250 ppm showed a reduction in body weight, compared to control animals, throughout the study. Food consumption in both sexes was reduced during the greater part of the study when compared to control values and food utilization in females was less efficient during week 1-4 of the study compared to control values. There was an increase in liver weight, after adjustment for terminal body weight, in both sexes at this dose level and there were some changes in clinical chemistry parameters. However there were no changes in absolute liver weight or liver histopathology and therefore these changes are considered to be of no toxicological significance. There were small perturbations in some red blood cell parameters in both sexes but these were considered to be of no toxicological significance. The changes at 500 ppm were in males only. There was an increase in liver weight after adjustment terminal body weight, a small reduction in plasma alkaline phosphatase activity and small perturbations in some red blood cell parameters.   There were no treatment-related effects in animals given 100 ppm. In conclusion, oral administration of 1250 ppm for 90 consecutive days produced signs of toxicity as evidenced by decreases in body weight and food consumption/utilization efficiency. Oral administration of 500 ppm produced no effects that were considered toxicologically significant. 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
94.4% purity
Species:
dog
Strain:
Beagle
Details on species / strain selection:
The dog is the preferred non-rodent species for regulatory studies and the Alderley Park beagle was used because of the substantial background data available for this strain, in the testing facility, relating to studies of this type.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 16-24 weeks old on arrival
- Housing: Dogs were housed by treatment group (sexes separately) in indoor pens. The pens have a sleeping platform (with a heated floor underneath) and interlinking gates which enable the dogs to be separated for feeding and dosing. Dogs received exercise (while on a lead) for all routine procedures, e.g. weighing, and were allowed free exercise weekly, on a group and sex basis, in an exercise area.
- Diet (e.g. ad libitum): Each morning, male dogs received 350 g and female dogs received 300 g of Laboratory Diet A, an expanded dry diet. During the pre-experimental period, food bowls were removed one hour after feeding to encourage the dogs to eat the diet quickly.
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 4°C
- Humidity (%): 40-70%
- Air changes (per hr): approximately 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route of administration:
oral: feed
Details on route of administration:
The oral route was chosen for administration of the test substance as this represents a possible route of exposure in humans and other mammalian species.
Vehicle:
other: Laboratory Diet A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted by soxhlet extraction with acetonitrile and portions of resulting solutions were diluted with acetonitrile, as appropriate, to give sample solution concentrations within the range of the calibration standards. These were analyzed by High Performance Liquid Chromatography (HPLC).

Nominally 100 mg of the test substance was accurately weighed into a volumetric flask and diluted to volume with acetonitrile (nominally 1.0 mg/mL). Further appropriate dilutions were made with acetonitrile in volumetric flasks to give a range of solutions, nominally within 5 μg/mL to 40 μg/mL. The purity of the test substance was not taken into account in the preparation of the standard solutions.

Accurately weighed portions of diet samples, 10 g, were transferred to extraction thimbles, 100 mL of acetonitrile was added to round bottom flasks and connected to soxhlet apparatus. The samples were extracted at a medium heat setting for 3 hours. The samples were diluted, as required, with acetonitrile to a known nominal concentration within the range of the calibration standards.

The following High Performance Liquid Chromatography Conditions were used: Pump: 600 Series (Waters); Mobile phase: Acetonitrile (325 mL), Water (175 mL), and Phosphoric acid (0.5 mL); Flow rate: l.5 mL/min; Detector: SA6500 or SA6504 (Servern Analytical); Detector wavelength: 245 nm; Column: 25 cm x 4.6 mm ID Hypersil BDS, (Shandon) or 15 cm x 3.9 mm ID Symmetry Cl8, (Waters); Column temperature: Ambient;
Sample introduction: 717 plus (Waters); Injection volume: 20 or25 μL; and Data handling: Waters 860 Data System (Waters).

The analysis system was calibrated using a range of standards to determine the linearity of response. An appropriate standard of known concentration was interspersed at intervals throughout the analysis.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
125 ppm
Dose / conc.:
250 ppm
Dose / conc.:
500 ppm
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: The dose levels selected for this study were based on the results of a preliminary oral dose ranging study involving 1 dog/sex/group, carried out in the testing facility.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least 3 times daily for clinical behavioural abnormalities (at dosing, after dosing and at the end of the working day). Individual daily assessment of faecal consistency were made for up to 5 hours post dosing: any subsequent assessments were made on a group basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; all dogs were also given a full clinical examination by a veterinarian pre-study and prior to termination. The examination included cardiac and pulmonary auscultation and indirect ophthalmoscopy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, before feeding, throughout the pre-study period, on day 1 and thereafter at weekly intervals, until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Food residues were recorded daily. During the pre-experimental period, food residues were recorded at approximately 1 hour after feeding. During the experimental period, food residues were recorded at approximately 4 hours after feeding. Any residual food was discarded. Food consumption was measured for at least 2 weeks pre-study and throughout the treatment period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-study and prior to termination
- Dose groups that were examined: all dogs were examined by indirect ophthalmoscopy

HAEMATOLOGY: Yes
- Time schedule for collection of blood: -1, 4, 8 weeks and prior to termination
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white cell count (WBC), differential WBC count, platelet count. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: -1, 4, 8 weeks and prior to termination
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: urea, glucose, total protein, potassium, calcium, total bilirubin, alkaline phosphatase activity, gamma-glutamyl transferase activity, creatinine, albumin, sodium, chloride, phosphorus (as phosphate), aspartate aminotransferase activity, alanine aminotransferase activity, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table). Organs weighed included adrenal glands, brain, kidneys, liver, ovaries, testes, thyroid glands. The left and right components of paired organs were weighed separately.

HISTOPATHOLOGY: Yes (see table). All tissues were submitted for histology except femur and stifle, epididymis, mammary gland, prostate gland, skin, trachea and voluntary muscle, which were stored. Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5µm and stained with haematoxylin and eosin. All selected tissues, together with abnormalities were examined by light microscopy.

From all animals at scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed: Adrenal glands, brain, ovaries, testes, kidneys, thyroid glands, and liver. The left and right components of paired organs were weighed separately.

The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative: Abnormal tissue, adrenal gland, aorta (abdominal), bone (femur and stifle joint), bone marrow (sternum), brain (cerebrum, cerebellum and brain stem), caecum, cervix with vagina, colon, duodenum, epididymis, eye, gall bladder, heart, ileum, Jejunum, kidney, liver, lung, lymph node – mesenteric, lymph node – prescapular, mammary gland (females) only, esophagus, ovary, oviduct, pancreas, parathyroid gland, pituitary gland, prostate gland, rectum, salivary gland – submandibular, sciatic nerve, skin (lateral thigh), spinal cord (cervical, thoracic and lumbar), spleen, sternum, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, uterus, and voluntary muscle (semimembranosus).

All tissues were submitted for histology except femur and stifle, epididymis, mammary gland, prostate gland, skin, trachea and voluntary muscle, which were stored. Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with hematoxylin and eosin.

All selected tissues, together with abnormalities were examined by light microscopy.
Statistics:
Bodyweights were considered by analysis of covariance on initial (week 1) bodyweight, separately for males and females. Weekly food consumption was considered by analysis of variance, separately for males and females. Haematology and blood clinical chemistry were considered by analysis of covariance on pre-experimental values. Male and female data were analyzed together and the results examined to determine whether any differences between control and treated groups were consistent between sexes. The covariate adjustment was based on the separate sex pre-experimental group means. Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females. The data from paired organs were examined for differential effects on left and right components. Summary data are also presented for organ to bodyweight ratios but these were not analyzed statistically as the analysis of covariance provides a better method of allowing for differences in terminal bodyweights. Analyses of variance and covariance, with the exception of organ weights, allowed for the replicate structure of the study design, and were carried out using the GLM procedure in SAS (1989). Least-squares means for each group were calculated using the LSMEAN option in SAS PROC GLM. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least-squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Dietary administration of the test substance produced no adverse clinical signs, although a slightly increased incidence of fluid faeces in males and of salivation at dosing in females was seen at a dose level of 500 ppm but was considered to be of no toxicological importance. The only clinical sign seen that was considered possibly to be related to treatment was a slightly increased incidence of salivation at dosing for females receiving 500 ppm.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related effects on bodyweight were seen for males receiving 250 ppm and for males and females receiving 500 ppm of the test substance. During week 1, there was a slight loss of bodyweight for all animals receiving 500 ppm. From weeks 2 to 14, group mean bodyweight, adjusted for initial weight, for males and females receiving 500 ppm was lower (up to approximately 7% lower) than that of concurrent controls, with these differences usually attaining statistical significance.

Group mean bodyweight, adjusted for initial weight, for males receiving 250 ppm was also lower (up to approximately 5% lower) than that of concurrent controls throughout weeks 5 to 14, although these differences did not usually attain statistical significance.

For males and females receiving 125 ppm, and for females receiving 250 ppm, group mean bodyweight, adjusted for initial weight, was similar to that of controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment-related effects on food consumption were seen for males and females receiving 500 ppm of the test substance. Throughout most weeks of the study, group mean food consumption for males and females receiving 500 ppm was slightly reduced, in comparison with that of concurrent controls, with these differences usually attaining statistical significance.

For animals receiving 125 or 250 ppm, there were no treatment-related effects on food consumption. Group mean food consumption for males and females receiving 250 ppm was marginally lower than that of concurrent controls during week 1. However, as individual food consumption values for these animals were similar to those seen during the pre-experimental period, this was considered to be unrelated to administration of the test substance
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no veterinary or ophthalmoscopy findings that were considered to be related to administration of the test substance.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related haematology changes were seen for males receiving 250 or 500 ppm of the test substance. Group mean platelet counts, adjusted for pre-experimental values, were slightly raised throughout the study for males receiving 500 ppm and during week 4 and 8 for males receiving 250 ppm. There were no changes in any of the other haematology parameters measured that could be attributed to administration of of the test substance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
During weeks 4, 8 and 13, group mean plasma albumin and/or total protein levels, adjusted for pre-experimental values were slightly reduced for males and females receiving 500 ppm, in comparison with concurrent control values. These differences usually attained statistical significance.

There were no other changes in any of the other blood clinical chemistry parameters measured that could be attributed to administration of the test substance.
Urinalysis findings:
not examined
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):
There was no evidence of any differential effects on left and right components of paired organs, with the exception of the kidney. Group mean left, right and combined kidney weights, adjusted for terminal bodyweight, for males receiving 500 ppm of the test substance were marginally higher (combined weights were approximately 12% higher) than that of concurrent controls. However, these differences only attained statistical significance for the right kidney weight. There was no evidence of any changes in any of the other organ weights measured.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Minimal focal unilateral tubular dilatation with eosinophilic casts was observed in the kidneys in 3/4 females which received 500 ppm of the test substance. Minimal focal unilateral tubular dilatation with eosinophilic casts was also observed in the kidneys in 1/4 males which received 250 ppm and in 1/4 males which received 500 ppm of the test substance. However, as single incidences of this finding have been seen historically in male and female dogs on 90 day studies within the test facility, and as no dose-related increase in incidence was apparent in males on this study, these isolated findings could not be attributed to administration of the test substance. There were no other microscopic findings considered to be related to administration of the test substance.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Key result
Critical effects observed:
no
Conclusions:
Dietary administration of the test substance at an inclusion level of 500 ppm produced reduced growth, reduced food consumption and slight reductions in plasma albumin and/or total protein levels in males and/or females. Reduced growth, without any adverse effects on food consumption, was also seen for males receiving 250 ppm of the test substance. There were no organ weight changes or histopathological findings that were considered to be of toxicological importance. A dietary inclusion level of 125 ppm of the test substance was without toxicologically significant effects in males, following daily oral administration for at least 90 days. For females, a dietary inclusion level of 250 ppm was without toxicologically significant effects, following daily oral administration for at least 90 days.
Executive summary:

Groups of 4 male and 4 female beagle dogs were fed diets containing 0, 125, 250, or 500 ppm of the test substance for 90 days. Dietary administration of the test substance produced no adverse clinical signs, although a slightly increased incidence of fluid faeces in males and of salivation at dosing in females was seen at a dose level of 500 ppm but was considered to be of no toxicological importance. Administration of the test substance at a dietary inclusion level of 500 ppm produced an initial bodyweight loss and slight inappetence during week 1. Bodyweight and food consumption for these animals remained slightly lower than those of controls throughout the remainder of the study and this was associated with slight reductions in plasma albumin and/or total protein levels. Bodyweight for males receiving 250 ppm of the test substance was also slightly lower than that of controls, although this was not associated with any treatment-related effects on food consumption. Slight increases in platelet counts were seen for males receiving 250 or 500 ppm of the test substance, but these effects were considered to be too small to be of toxicological importance. At necropsy, marginally increased kidney weight was seen for males receiving 500 ppm of the test substance but, as this was not associated with any treatment-related histopathological changes, this was considered to be of no toxicological importance. Tubular dilatation with eosinophilic casts was observed in the kidneys in 3/4 females which received 500 ppm of the test substance. However, this histopathological change was minimal, focal and unilateral and was considered to be of no toxicological importance. A dietary inclusion level of 125 ppm of the test substance was without toxicologically significant effects in males, following daily oral administration for at least 90 days. For females, a dietary inclusion level of 250 ppm was without toxicologically significant effects, following daily oral administration for at least 90 days.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
4.6 mg/kg bw/day
Study duration:
chronic
Species:
dog

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Necropsy, blood collection, urine collection and body weight was performed on day 30 for G1, G2 and G3 instead of day 29.
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean of 184.6-204 g (males); mean of 146.8-159.0 g (females)
- Housing: Rats were housed in standard polypropylene cages group wise and sex wise, each cage containing three rats for range finding study and five rats for main study. Enrichment materials (Tunnels and Crawl balls) were used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 7 days; rats were acclimatized to the restraint tubes for a short period during the acclimation period prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.8°C
- Humidity (%): 43.2 – 61.5%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 1 - < 3 µm
Geometric standard deviation (GSD):
1
Remarks on MMAD:
Aerosol size measurements were collected in the pilot study to confirm respirability.
Details on inhalation exposure:
Prior to the main study, a range finding study where 3 male and 3 female rats were exposed nose only to 0.50 mg/L of the test substance was performed. The 0.50 mg/L target concentration was based upon a previously reported LC50 values of >2.12 mg/L for male and 3.19 mg/L for female rats. Two females exposed to 0.50 mg/L died within 2 hours of exposure while all other animals were euthanized without necropsy on day 3 post-exposure. A second range finding study was performed to select the tolerable concentration levels for the repeated exposure study. Three male and three female rats were exposed to 0.025 mg/L of the test substance, 6 hours/day, for five days with two additional days of observation. All the rats tolerated the 0.025 mg/L exposure level for five days. Therefore, the target concentration levels of 0.001, 0.01 and 0.025 mg/L were selected for the low, intermediate and high concentration groups, respectively for the repeated exposure main study.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hours a day, 5 days a week
Frequency of treatment:
4 weeks
Dose / conc.:
0.001 mg/L air (nominal)
Dose / conc.:
0.01 mg/L air (nominal)
Dose / conc.:
0.025 mg/L air (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor.
Positive control:
No
Observations and examinations performed and frequency:
Six groups were included in this study: Group G1 was the control group; Group G2 was the low-dose group (0.001 mg/L); Group G3 was the intermediate-dose group (0.01 mg/L); Group G4 was the high-dose group (0.025 mg/L); Group G5 was the Sattelite group control; and Group G6 was the Exposed Satellite group (0.01 mg/L).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the rats were observed individually during and after the exposure and daily throughout the experimental period. Cage-side observations included changes in the skin and fur, eyes, and mucous membranes; changes in respiratory and circulatory systems, changes in nervous system, and changes in somatomotor activity and behavior patterns. Attention was directed to observation of tremors, convulsions, salivation, diahorrea, lethargy (dullness), sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During and after exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were measured on day 0, 3, 7, 10, 14, 17, 21, 24, 28, 29 and 30. The satellite group (G5 and G6) of animals were weighed additionally on day 31, 35, 38, 42 and 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; cage wise feed consumption was recorded daily and reported weekly.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: cage wise water consumption was recorded daily and reported weekly.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All
- Parameters examined: RBC count, WBC count, hematocrit (HCT, PCV), platelet count, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), differential count (5 parts) using a Hematology Bayer Advia 120 fully automated analyzer. Prothrombin time was measured by using CoaData 2001 coagulometer. Reticulocyte count and clotting time were done by manual method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Glucose, total cholesterol, triglycerides, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP, SAP), blood urea nitrogen (BUN), urea, creatinine, total protein, albumin, globulin, phosphorus and calcium levels were analysed using Siemens Dimension Xpand fully automated biochemistry analyzer. Sodium, chloride and potassium were analyzed by Human Humalyte electrolyte analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: 1. Color – Visual observation 2. pH – Multistrip 3.Specific gravity – Multistrip 4. Sediment – Visual observation 5. Albumin – Multistrip 6.Glucose – Multistrip 7. Bilirubin – Multistrip 8.Ketones – Acetone – Multistrip 9.Blood – Multistrip 10.WBC – Microscopic 11. RBC– Microscopic 12.Epithelial cells– Microscopic 13.Casts– Microscopic 14.Crystals– Microscopic 15.Organisms– Microscopic 16.Abnormal constituents – Microscopic.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following organs were collected and weighed wet as soon as possible after dissection: Brain, Heart, Lungs, Liver, Kidneys, Epididymides, Spleen, Thymus, Adrenals, Testes, Ovaries, and Uterus.

The following organs/tissues were collected from all animals and preserved in 10% neutral buffered formalin. Testes and eyes will be pre-fixed with Modified Davidson’s and Davidson’s fixative, respectively for 48 hours, washed in running tap water for 5 minutes and were transferred into 10% neutral buffered formalin. The lungs were removed intact, weighed as mentioned above and inflated with 10% neutral buffered formalin: Heart & Aorta, Spleen, Prostate, Nasopharyngeal tissues, Liver, Thymus, Epididymides, Trachea, Kidneys, Brain, Mammary Gland, Lungs, Pituitary, Larynx, Lacryminal gland, Lymph nodes (Mediastinal, Maxillary, Mesenteric, and Tracheobronchial), Sciatic nerve, Eyes, Salivary gland, Adrenals, Testes, Esophagus, Stomach, Ovaries, Skin, Small Intestine, Uterus, Thyroid with parathyroid, Bone marrow (sternum), Large intestine, Pancreas, Urinary bladder, Spinal cord (cervical, mid-thoracic, and lumbar), and Seminal vesicles.

All test animals were subjected to a detailed gross pathological examination. The tissues from all the groups in the above list were subjected for histopathology examination. These organ/tissue samples were processed, embedded with paraffin wax, sectioned at approximately 5 μm thick and stained with hematoxylin and eosin. The histological sections of the respiratory tract were taken as follows: Nasopharyngeal tissues (Four levels: posterior upper incisor, incisive papilla, second palatine crest, and first molar teeth levels), Trachea (Two levels: Transverse and Longitudinal horizontal), Larynx (Three Levels: Base of epiglottis, Ventral pouch, and Cricoid cartilage), and Lungs (Sections of all five lobes).
Statistics:
The data was subjected to Modified-Levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA) followed by Student-Newman-Keul’s test for post hoc comparison. Heterogeneous data was subjected to non-parametric tests (NCSS 2007 statistical software). Data for group 2 (0.001 mg/L), group 3 (0.01 mg/L) were compared to the group 1 air-exposed control group. Data for the group 4 (0.025 mg/L) and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group. The organ weight data for groups 2 (0.001 mg/L), group 3 (0.01 mg/L) and 4 (0.025 mg/L) was compared to group 1 air-exposed control group and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats exposed to 0.0017 mg/L (low-dose) and 0.025 mg/L (high-dose) demonstrated statistically significant increases in body weights when compared to the control on test days 10 and 28, respectively. Since these changes were increases and not decreases they were considered spurious, non-adverse and not test substance-related. Female rats exposed to 0.012 mg/L (satellite group) demonstrated statistically significant increase in body weights on test days 21, 28, 29, 31 and 35 when compared to the respective control group values and were considered to be non-adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no exposure-related differences in haematology or coagulation parameters in male or female rats following the treatment or recovery periods.

Mean corpuscular hemoglobin (MCH) was minimally decreased at test day 29 in high-dose females. A decrease in reticulocyte count (RC) was also seen at test day 29 in high-dose group females. The minimal decrease in MCH and RC occurred in the absence of clear correlation with other red cell indices. Therefore, this minimal decrease in MCH and RC was considered unrelated to treatment and non-adverse.

Clotting time was minimally decreased in high-dose group females at test day 29. Similar change in clotting time was not observed in male rats. This change was likely spurious and unrelated to treatment. In addition minimal decrease in clotting time has no toxicologic significance; therefore, this change was considered to be non-adverse.

The following statistically significant changes in mean haematology or coagulation parameters were considered unrelated to treatment because they either only occurred during pretest or were not dose related: On day 0 (Pre-test), there were an increases in the eosinophils and MCV and decreases in the clotting time and WBC counts of high-dose group females when compared to the satellite Control group; On day 30, males of low-dose group showed an increase in clotting time when compared to the control group; On day 29, satellite group females showed an increase in monocytes when compared to the satellite control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they only occurred during pretest: On day 0 (Pre-test), an increase in potassium levels of satellite group males when compared to the satellite control group.

The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they were not dose related or individual values in these groups were in range of the historical control data: On day 29, an increase in glucose, calcium and sodium concentration were observed in the high-dose group males when compared to the satellite control group. These parameters were well within the historical control data. Current historical range in males for glucose is 77 - 126 mg/dL, for calcium is 7.9 - 12.7 mg/dL and for sodium is 139 - 149 mmol/L; On day 29, an increase in the glucose levels were observed in the high-dose group females when compared to the satellite control group. However, the mean was within the current historical range (74 - 127 mg/dL).

The following statistically significant change in group mean biochemical parameters in the two-week recovery groups were unrelated to treatment because similar changes were not observed at the end of the 28-day exposure period: A decrease in phosphorus and potassium levels of satellite group males was observed when compared to the satellite control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in group mean urinalyses parameters in male or female rats following the treatment or recovery periods.
Urinalysis performed on day 0, days 29/30 and day 43 did not show any treatment-related changes in any of the groups. The physical parameters like color, appearance, specific gravity, sediment, pH or biochemical parameters like glucose, albumin, bilirubin, ketone bodies and the microscopic parameters like blood, leukocytes, erythrocytes, epithelial cells, casts and crystals in treated groups were comparable with control animals and were well within the historical control data. No abnormal consituents or organisms were observed in the urinalysis.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related organ weight changes were observed in the treated animals.

The following statistical differences observed in the absolute and relative organ weights of treated animals were considered incidental and unrelated to exposure of test substance because they were unaccompanied by correlated morphologic findings or did not occur in dose dependent manner or were seen only in one sex: an increase in mean absolute and mean relative (%body weight) liver weights in G3 males (0.01 mg/L); an increase in mean absolute liver weights in G3 females (0.01 mg/L); an increase in mean absolute and mean relative (%body weight) epididymides weights in G2 (0.001 mg/L) and G3 (0.01 mg/L) males; an increase in mean absolute spleen weight in G3 males (0.01 mg/L); a decrease in mean absolute heart and adrenals weight in G4 males (0.025 mg/L); and an increase in mean relative (%body weight) brain weight in G4 females (0.025 mg/L).

In recovery groups, statistical differences were observed in some absolute and relative organ weights of treated animals as compared to recovery controls. These organ weight differences in the recovery animals were interpreted to be spurious and unrelated to exposure of test substance since organ weight effects were observed only in one sex or not observed in the main group males or females. These included: a decrease in mean absolute and relative (%body weight) spleen weights in females; an increase in relative (%body weight) brain weight in males and decreased relative (%body weight) brain weight in females and an increase in mean absolute uterus weight in females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related gross pathological findings were observed in treated animals. The gross findings observed in this study were consistent with spontaneous findings of the type routinely observed in Wistar rats of this age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related histopathological findings were observed in exposed animals.

A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. In normal mature rats, the outer nuclear layer (ONL) in the mid-posterior region of the retina generally consists of 10-11 photoreceptor nuclei. In the mild retinal degeneration observed, the ONL was decreased to 6-9 nuclei, but the retinal layer architecture appeared normal except for some pyknotic nuclei in the inner nuclear layer. With increased in severity of retinal degeneration, the width of the ONL was further decreased and the photoreceptor segments were degenerated. When the outer retinal layers were degenerated to the 0-1 layer, the inner retina was abutted directly on the retinal pigment epithelium (RPE). Other histopathology changes in the retina included pyknotic nuclei in the inner nuclear layer. With few exceptions, inner retinal layers (nerve fiber layer, ganglion cell layers, and inner plexiform layers) were relatively unaffected, even in cases of observed marked retinal degeneration. Retinal degeneration was observed mostly in the mid-posterior region, while changes in the mid-peripheral region were minimal.

The histopathology changes observed in retina were not considered to be test substance related effects based on the following observations:

● Retinal lesions were observed in all groups (except G2), including controls, and in all rats in the control recovery groups in this study.
● In previous chronic oral study with the same test material, no test substance related retinal changes were present at any dose.

In conclusion, the retinal lesions observed in this study were not considered to be indicative of target organ toxicity to the eye. All other histopathology findings observed in this study were interpreted to be spontaneous changes of the type routinely observed in Wistar rats of this age.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Rats from the low target concentration (0.001 mg/L) group (G2) were exposed to 0.0017 ± 0.00022 mg/L of the test substance (Mean ± SD) which was characterized by a MMAD (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration (0.01 mg/L) group (G3) were exposed to 0.012 ± 0.0025 mg/L of the test substance with a MMAD (GSD) of 2.15 μm (2.77). Rats from the high target concentration (0.025 mg/L) group (G4) were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD (GSD) of 2.12 μm (2.79). Rats from the intermediate-satellite target concentration (0.01 mg/L) group (G6) were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD (GSD) of 2.61 μm (2.84).
Key result
Dose descriptor:
NOAEC
Effect level:
0.025 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest concentration tested
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.
Executive summary:

Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the of the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor. Animals were observed for clinical signs of toxicity daily, had body weights measured twice weekly and feed and water consumption determined daily and reported weekly. Three to four days following the final exposure the air-control, low, intermediate and high concentrations groups of 10 rats (5 male and 5 female) each were fasted overnight, blood and urine samples collected and sacrificed for anatomic pathology evaluation including microscopic tissue evaluation. Three days following exposure the air (control) satellite and the intermediate satellite groups of 10 rats (5 male and 5 females) each were fasted overnight, blood and urine samples collected. Two weeks following exposure the air (control) satellite and the intermediate satellite groups of 10 rats each were fasted overnight, blood and urine samples collected and were sacrificed for anatomic pathology evaluation including microscopic tissue evaluation.

 

Rats from the low target concentration group were exposed to a gravimetrically determined aerosol concentration of 0.0017 ± 0.00022 mg/L of the test substance (mean ± SD), which was characterized by a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration group were exposed to 0.012 ± 0.0039 mg/L of the test substance with a MMAD of 2.15 μm (2.77). Rats from the high target concentration group were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD of 2.12 μm (2.79). Rats from the intermediate satellite target concentration group were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD of 2.61 μm (2.84).  

Exposure to the test substance did not result in test substance related adverse changes in body weight, weekly feed and water consumption, and no clinical signs of toxicity were observed during the course of this study. There were no exposure-related differences in haematology or coagulation, clinical biochemistry parameters in male or female rats following the treatment or recovery periods.  

There were no test substance-related organ weight changes or gross pathological changes observed in this study. No test-substance related changes in microscopic histopathology were observed in this study. A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. The microscopic changes observed in the retina were not considered to be test substance related effects based on the following observations: Retinal lesions were observed in all groups (except at 0.001 mg/L), including controls, and in all rats in the control recovery/satellite groups in this study. In addition, in a previous 2 year chronic study with the test substance, no test substance related retinal changes were present at any dose in rats.  

Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Necropsy, blood collection, urine collection and body weight was performed on day 30 for G1, G2 and G3 instead of day 29.
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean of 184.6-204 g (males); mean of 146.8-159.0 g (females)
- Housing: Rats were housed in standard polypropylene cages group wise and sex wise, each cage containing three rats for range finding study and five rats for main study. Enrichment materials (Tunnels and Crawl balls) were used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 7 days; rats were acclimatized to the restraint tubes for a short period during the acclimation period prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.8°C
- Humidity (%): 43.2 – 61.5%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 1 - < 3 µm
Geometric standard deviation (GSD):
1
Remarks on MMAD:
Aerosol size measurements were collected in the pilot study to confirm respirability.
Details on inhalation exposure:
Prior to the main study, a range finding study where 3 male and 3 female rats were exposed nose only to 0.50 mg/L of the test substance was performed. The 0.50 mg/L target concentration was based upon a previously reported LC50 values of >2.12 mg/L for male and 3.19 mg/L for female rats. Two females exposed to 0.50 mg/L died within 2 hours of exposure while all other animals were euthanized without necropsy on day 3 post-exposure. A second range finding study was performed to select the tolerable concentration levels for the repeated exposure study. Three male and three female rats were exposed to 0.025 mg/L of the test substance, 6 hours/day, for five days with two additional days of observation. All the rats tolerated the 0.025 mg/L exposure level for five days. Therefore, the target concentration levels of 0.001, 0.01 and 0.025 mg/L were selected for the low, intermediate and high concentration groups, respectively for the repeated exposure main study.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hours a day, 5 days a week
Frequency of treatment:
4 weeks
Dose / conc.:
0.001 mg/L air (nominal)
Dose / conc.:
0.01 mg/L air (nominal)
Dose / conc.:
0.025 mg/L air (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor.
Positive control:
No
Observations and examinations performed and frequency:
Six groups were included in this study: Group G1 was the control group; Group G2 was the low-dose group (0.001 mg/L); Group G3 was the intermediate-dose group (0.01 mg/L); Group G4 was the high-dose group (0.025 mg/L); Group G5 was the Sattelite group control; and Group G6 was the Exposed Satellite group (0.01 mg/L).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the rats were observed individually during and after the exposure and daily throughout the experimental period. Cage-side observations included changes in the skin and fur, eyes, and mucous membranes; changes in respiratory and circulatory systems, changes in nervous system, and changes in somatomotor activity and behavior patterns. Attention was directed to observation of tremors, convulsions, salivation, diahorrea, lethargy (dullness), sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During and after exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were measured on day 0, 3, 7, 10, 14, 17, 21, 24, 28, 29 and 30. The satellite group (G5 and G6) of animals were weighed additionally on day 31, 35, 38, 42 and 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; cage wise feed consumption was recorded daily and reported weekly.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: cage wise water consumption was recorded daily and reported weekly.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All
- Parameters examined: RBC count, WBC count, hematocrit (HCT, PCV), platelet count, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), differential count (5 parts) using a Hematology Bayer Advia 120 fully automated analyzer. Prothrombin time was measured by using CoaData 2001 coagulometer. Reticulocyte count and clotting time were done by manual method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Glucose, total cholesterol, triglycerides, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP, SAP), blood urea nitrogen (BUN), urea, creatinine, total protein, albumin, globulin, phosphorus and calcium levels were analysed using Siemens Dimension Xpand fully automated biochemistry analyzer. Sodium, chloride and potassium were analyzed by Human Humalyte electrolyte analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: 1. Color – Visual observation 2. pH – Multistrip 3.Specific gravity – Multistrip 4. Sediment – Visual observation 5. Albumin – Multistrip 6.Glucose – Multistrip 7. Bilirubin – Multistrip 8.Ketones – Acetone – Multistrip 9.Blood – Multistrip 10.WBC – Microscopic 11. RBC– Microscopic 12.Epithelial cells– Microscopic 13.Casts– Microscopic 14.Crystals– Microscopic 15.Organisms– Microscopic 16.Abnormal constituents – Microscopic.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following organs were collected and weighed wet as soon as possible after dissection: Brain, Heart, Lungs, Liver, Kidneys, Epididymides, Spleen, Thymus, Adrenals, Testes, Ovaries, and Uterus.

The following organs/tissues were collected from all animals and preserved in 10% neutral buffered formalin. Testes and eyes will be pre-fixed with Modified Davidson’s and Davidson’s fixative, respectively for 48 hours, washed in running tap water for 5 minutes and were transferred into 10% neutral buffered formalin. The lungs were removed intact, weighed as mentioned above and inflated with 10% neutral buffered formalin: Heart & Aorta, Spleen, Prostate, Nasopharyngeal tissues, Liver, Thymus, Epididymides, Trachea, Kidneys, Brain, Mammary Gland, Lungs, Pituitary, Larynx, Lacryminal gland, Lymph nodes (Mediastinal, Maxillary, Mesenteric, and Tracheobronchial), Sciatic nerve, Eyes, Salivary gland, Adrenals, Testes, Esophagus, Stomach, Ovaries, Skin, Small Intestine, Uterus, Thyroid with parathyroid, Bone marrow (sternum), Large intestine, Pancreas, Urinary bladder, Spinal cord (cervical, mid-thoracic, and lumbar), and Seminal vesicles.

All test animals were subjected to a detailed gross pathological examination. The tissues from all the groups in the above list were subjected for histopathology examination. These organ/tissue samples were processed, embedded with paraffin wax, sectioned at approximately 5 μm thick and stained with hematoxylin and eosin. The histological sections of the respiratory tract were taken as follows: Nasopharyngeal tissues (Four levels: posterior upper incisor, incisive papilla, second palatine crest, and first molar teeth levels), Trachea (Two levels: Transverse and Longitudinal horizontal), Larynx (Three Levels: Base of epiglottis, Ventral pouch, and Cricoid cartilage), and Lungs (Sections of all five lobes).
Statistics:
The data was subjected to Modified-Levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA) followed by Student-Newman-Keul’s test for post hoc comparison. Heterogeneous data was subjected to non-parametric tests (NCSS 2007 statistical software). Data for group 2 (0.001 mg/L), group 3 (0.01 mg/L) were compared to the group 1 air-exposed control group. Data for the group 4 (0.025 mg/L) and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group. The organ weight data for groups 2 (0.001 mg/L), group 3 (0.01 mg/L) and 4 (0.025 mg/L) was compared to group 1 air-exposed control group and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats exposed to 0.0017 mg/L (low-dose) and 0.025 mg/L (high-dose) demonstrated statistically significant increases in body weights when compared to the control on test days 10 and 28, respectively. Since these changes were increases and not decreases they were considered spurious, non-adverse and not test substance-related. Female rats exposed to 0.012 mg/L (satellite group) demonstrated statistically significant increase in body weights on test days 21, 28, 29, 31 and 35 when compared to the respective control group values and were considered to be non-adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no exposure-related differences in haematology or coagulation parameters in male or female rats following the treatment or recovery periods.

Mean corpuscular hemoglobin (MCH) was minimally decreased at test day 29 in high-dose females. A decrease in reticulocyte count (RC) was also seen at test day 29 in high-dose group females. The minimal decrease in MCH and RC occurred in the absence of clear correlation with other red cell indices. Therefore, this minimal decrease in MCH and RC was considered unrelated to treatment and non-adverse.

Clotting time was minimally decreased in high-dose group females at test day 29. Similar change in clotting time was not observed in male rats. This change was likely spurious and unrelated to treatment. In addition minimal decrease in clotting time has no toxicologic significance; therefore, this change was considered to be non-adverse.

The following statistically significant changes in mean haematology or coagulation parameters were considered unrelated to treatment because they either only occurred during pretest or were not dose related: On day 0 (Pre-test), there were an increases in the eosinophils and MCV and decreases in the clotting time and WBC counts of high-dose group females when compared to the satellite Control group; On day 30, males of low-dose group showed an increase in clotting time when compared to the control group; On day 29, satellite group females showed an increase in monocytes when compared to the satellite control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they only occurred during pretest: On day 0 (Pre-test), an increase in potassium levels of satellite group males when compared to the satellite control group.

The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they were not dose related or individual values in these groups were in range of the historical control data: On day 29, an increase in glucose, calcium and sodium concentration were observed in the high-dose group males when compared to the satellite control group. These parameters were well within the historical control data. Current historical range in males for glucose is 77 - 126 mg/dL, for calcium is 7.9 - 12.7 mg/dL and for sodium is 139 - 149 mmol/L; On day 29, an increase in the glucose levels were observed in the high-dose group females when compared to the satellite control group. However, the mean was within the current historical range (74 - 127 mg/dL).

The following statistically significant change in group mean biochemical parameters in the two-week recovery groups were unrelated to treatment because similar changes were not observed at the end of the 28-day exposure period: A decrease in phosphorus and potassium levels of satellite group males was observed when compared to the satellite control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in group mean urinalyses parameters in male or female rats following the treatment or recovery periods.
Urinalysis performed on day 0, days 29/30 and day 43 did not show any treatment-related changes in any of the groups. The physical parameters like color, appearance, specific gravity, sediment, pH or biochemical parameters like glucose, albumin, bilirubin, ketone bodies and the microscopic parameters like blood, leukocytes, erythrocytes, epithelial cells, casts and crystals in treated groups were comparable with control animals and were well within the historical control data. No abnormal consituents or organisms were observed in the urinalysis.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related organ weight changes were observed in the treated animals.

The following statistical differences observed in the absolute and relative organ weights of treated animals were considered incidental and unrelated to exposure of test substance because they were unaccompanied by correlated morphologic findings or did not occur in dose dependent manner or were seen only in one sex: an increase in mean absolute and mean relative (%body weight) liver weights in G3 males (0.01 mg/L); an increase in mean absolute liver weights in G3 females (0.01 mg/L); an increase in mean absolute and mean relative (%body weight) epididymides weights in G2 (0.001 mg/L) and G3 (0.01 mg/L) males; an increase in mean absolute spleen weight in G3 males (0.01 mg/L); a decrease in mean absolute heart and adrenals weight in G4 males (0.025 mg/L); and an increase in mean relative (%body weight) brain weight in G4 females (0.025 mg/L).

In recovery groups, statistical differences were observed in some absolute and relative organ weights of treated animals as compared to recovery controls. These organ weight differences in the recovery animals were interpreted to be spurious and unrelated to exposure of test substance since organ weight effects were observed only in one sex or not observed in the main group males or females. These included: a decrease in mean absolute and relative (%body weight) spleen weights in females; an increase in relative (%body weight) brain weight in males and decreased relative (%body weight) brain weight in females and an increase in mean absolute uterus weight in females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related gross pathological findings were observed in treated animals. The gross findings observed in this study were consistent with spontaneous findings of the type routinely observed in Wistar rats of this age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related histopathological findings were observed in exposed animals.

A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. In normal mature rats, the outer nuclear layer (ONL) in the mid-posterior region of the retina generally consists of 10-11 photoreceptor nuclei. In the mild retinal degeneration observed, the ONL was decreased to 6-9 nuclei, but the retinal layer architecture appeared normal except for some pyknotic nuclei in the inner nuclear layer. With increased in severity of retinal degeneration, the width of the ONL was further decreased and the photoreceptor segments were degenerated. When the outer retinal layers were degenerated to the 0-1 layer, the inner retina was abutted directly on the retinal pigment epithelium (RPE). Other histopathology changes in the retina included pyknotic nuclei in the inner nuclear layer. With few exceptions, inner retinal layers (nerve fiber layer, ganglion cell layers, and inner plexiform layers) were relatively unaffected, even in cases of observed marked retinal degeneration. Retinal degeneration was observed mostly in the mid-posterior region, while changes in the mid-peripheral region were minimal.

The histopathology changes observed in retina were not considered to be test substance related effects based on the following observations:

● Retinal lesions were observed in all groups (except G2), including controls, and in all rats in the control recovery groups in this study.
● In previous chronic oral study with the same test material, no test substance related retinal changes were present at any dose.

In conclusion, the retinal lesions observed in this study were not considered to be indicative of target organ toxicity to the eye. All other histopathology findings observed in this study were interpreted to be spontaneous changes of the type routinely observed in Wistar rats of this age.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Rats from the low target concentration (0.001 mg/L) group (G2) were exposed to 0.0017 ± 0.00022 mg/L of the test substance (Mean ± SD) which was characterized by a MMAD (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration (0.01 mg/L) group (G3) were exposed to 0.012 ± 0.0025 mg/L of the test substance with a MMAD (GSD) of 2.15 μm (2.77). Rats from the high target concentration (0.025 mg/L) group (G4) were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD (GSD) of 2.12 μm (2.79). Rats from the intermediate-satellite target concentration (0.01 mg/L) group (G6) were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD (GSD) of 2.61 μm (2.84).
Key result
Dose descriptor:
NOAEC
Effect level:
0.025 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest concentration tested
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.
Executive summary:

Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the of the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor. Animals were observed for clinical signs of toxicity daily, had body weights measured twice weekly and feed and water consumption determined daily and reported weekly. Three to four days following the final exposure the air-control, low, intermediate and high concentrations groups of 10 rats (5 male and 5 female) each were fasted overnight, blood and urine samples collected and sacrificed for anatomic pathology evaluation including microscopic tissue evaluation. Three days following exposure the air (control) satellite and the intermediate satellite groups of 10 rats (5 male and 5 females) each were fasted overnight, blood and urine samples collected. Two weeks following exposure the air (control) satellite and the intermediate satellite groups of 10 rats each were fasted overnight, blood and urine samples collected and were sacrificed for anatomic pathology evaluation including microscopic tissue evaluation.

 

Rats from the low target concentration group were exposed to a gravimetrically determined aerosol concentration of 0.0017 ± 0.00022 mg/L of the test substance (mean ± SD), which was characterized by a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration group were exposed to 0.012 ± 0.0039 mg/L of the test substance with a MMAD of 2.15 μm (2.77). Rats from the high target concentration group were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD of 2.12 μm (2.79). Rats from the intermediate satellite target concentration group were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD of 2.61 μm (2.84).  

Exposure to the test substance did not result in test substance related adverse changes in body weight, weekly feed and water consumption, and no clinical signs of toxicity were observed during the course of this study. There were no exposure-related differences in haematology or coagulation, clinical biochemistry parameters in male or female rats following the treatment or recovery periods.  

There were no test substance-related organ weight changes or gross pathological changes observed in this study. No test-substance related changes in microscopic histopathology were observed in this study. A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. The microscopic changes observed in the retina were not considered to be test substance related effects based on the following observations: Retinal lesions were observed in all groups (except at 0.001 mg/L), including controls, and in all rats in the control recovery/satellite groups in this study. In addition, in a previous 2 year chronic study with the test substance, no test substance related retinal changes were present at any dose in rats.  

Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 58 days (males); approximately 59 days (females)
- Weight at study initiation: 255.1-294.7 g (males); 174.9-221.1 g (females)
- Housing: Animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Enrichment (nylabone toy) was placed in each cage. Each cage rack contained only animals of one sex.
.- Diet (e.g. ad libitum): ad libitum, except when rats were fasted
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
The test material was moistened with water to form a thick paste
Details on exposure:
Approximately 24 hours prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. The target area to be treated (5 cm x 7.4 cm = 37 cm2) was marked on the back of each rat with a water-insoluble marker. The 37 cm2 treatment area is approximately equal to 10% of the total body surface area for rats in the 200 to 300 g range of body weights.

The test substance was moistened with deionized water to form a thick paste and applied in a thin and uniform layer to cover as much of surface of the target area as possible. The test site was covered with a 2-ply porous gauze pad followed by successive layers of stretch gauze (no more than 8 layers) and self-adhesive bandage. The control rats were treated with deionized water at the same volume as the high-dose rats. Some rats were fitted with plastic collars during the exposure period to prevent disruption of the wrappings. The rats were checked for dislodged wrappings several times daily during the exposure period.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 0, 100, 300, and 1000 mg/kg/day were selected for this study. The 1000 mg/kg/day dosage is a limit dose level. The other dosages were selected to establish a no-observed-adverse-effect level (NOAEL) and to assess a dose response for any observed effects.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least twice daily throughout the study. Abnormal behavior/appearance was recorded by exception.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before dosing on test day 0 and then weekly thereafter soon after test substance removal, each rat was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed on test day 0 and at weekly intervals during the study. All rats were weighed on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; The amount of food consumed by each rat was determined weekly by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. Food spillage < 5 g was not included in the calculation. From these measurements, mean daily food consumption over the interval was determined.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes; calculated from food consumption and body weight data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes; Two ophthalmology examinations were conducted by a veterinary ophthalmologist. The pretest examination was performed on all rats received for the study, prior to assignment to groups. Two rats with preexisting ophthalmology abnormalities were eliminated from consideration for use in the study. Surviving rats were examined prior to the final sacrifice. Both eyes of each rat were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes; carbon dioxide
- Animals fasted: Yes
- How many animals: All
- Parameters examined: red blood cell count, red cell distribution width, hemoglobin, absolute reticulocyte count, hematocrit, platelet count, mean corpuscular (cell) volume, white blood cell count, mean corpuscular (cell) hemoglobin, differential white blood cell count, mean corpuscular (cell) hemoglobin concentration, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes
- How many animals: All
- Parameters examined: aspartate aminotransferase, glucose, alanine aminotransferase, total protein, sorbitol dehydrogenase albumin, alkaline phosphatase, globulin, total bilirubin, calcium, urea nitrogen, inorganic phosphorus, creatinine, sodium, cholesterol, potassium, triglycerides, and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: quality, ketone, color, bilirubin, clarity, blood, volume, urobilinogen, specific gravity (SG), protein, pH, microscopic urine sediment examination, and glucose.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following tissues were collected from all of the rats:
Cardiovascular System: Heart and aorta
Digestive System: Liver, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum, salivary glands, and pancreas
Endocrine System: Pituitary gland, thyroid gland, parathyroid glands, and adrenal glands
Female Reproductive System: Ovaries (with oviducts), uterus (including cervix), mammary glands, and vagina
Hematopoietic System; Spleen, thymus, mandibular lymph node, mesenteric lymph node, bone marrow, and Peyer’s patches
Male Reproductive System: Testes, epididymides, prostate, and seminal vesicles with coagulating glands
Miscellaneous: Skin (treated and adjacent material), skin adjacent to the mammary gland, eyes (including retina and optic nerves), and gross observations
Musculoskeletal System: Skeletal muscle, femur/knee joint, and sternum
Nervous System: Brain (including cerebrum, midbrain, cerebellum, and medulla/pons), spinal cord (cervical, mid-thoracic, and lumbar sections), and sciatic nerve
Respiratory System: Lungs, trachea, nose (4 sections), larynx, and pharynx
Urinary System: Kidneys and urinary bladder

The following tissues were weighed from rats sacrificed by design at the end of the study: liver, kidneys, heart, spleen, thymus, adrenal glands, brain, testes, epididymides, accessory sex organs (prostate + seminal vesicles with coagulating glands), ovaries (with oviducts) and uterus (including cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated. Testes, epididymides, and eyes were fixed in modified Davidson’s solution. All other tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist. All collected tissues from rats in the control and 1000 mg/kg/day groups, and tissues collected from early decedents were processed to slides and evaluated microscopically.
Statistics:
For Body Weight, Body Weight Gain, Food Consumption, Food Efficiency, Clinical pathology,and Organ weights, use Levene’s test for homogeneity and the Shapiro-Wilk test for normality; If the preliminary test is not significant, use the One-way analysis of variance test followed by Dunnett’s test; If the preliminary test is significant, use the Kruskal-Wallis test followed by Dunn’s test. Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of systemic toxicity were observed. Incidental incidences of ocular or nasal discharge, scabs, wounds, wet fur, paleness, and stained skin/fur were not considered to be test substance related as the clinical signs were observed in both treated and control animals and/or the incidence did not occur in a dose-related pattern. Swollen head or nose observed in several animals was considered to be due to the wrapping procedure.
Dermal irritation:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the course of the study, one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group died on test days 14, 9, 9, and 13, respectively. The deaths of these rats were considered to be a result of the wrappings being too tight.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effect was observed on mean body weight in any male or female group. Mean body weights on test day 28 in the 1000 mg/kg/day groups were 101% and 98% of control (neither statistically significant) for males and females, respectively.

No adverse or test substance-related effect was observed on mean body weight gain in any male or female group. Mean overall (test days 0-28) body weight gain in the 1000 mg/kg/day male group was 101% of control (not statistically significant). Mean overall body weight gain in the 1000 mg/kg/day female group was 80% of control. This decrease in body weight gain was considered to be non-adverse because the decrease was not statistically significant compared to control and was driven by the low body weight gain of one animal.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related effect was observed on mean overall food consumption (test days 0-28) in any male or female group. The mean overall food consumption in the 1000 mg/kg/day groups was 102% and 99% of control (neither statistically significant) for males and females, respectively.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse or test substance-related effect was observed on mean overall food efficiency (test days 0-28) in any male or female group. The mean overall food efficiency in the 1000 mg/kg/day male group was 99% of control (not statistically significant). The mean overall food efficiency in the 1000 mg/kg/day female group was 83% of control (not statistically significant). This decrease correlated with the decreased body weight gain in that group and was considered to be non-adverse.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological changes were noted in male or female rats at any dose level.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or adverse changes in group mean haematology parameters at test day 29 in male or female rats. The following statistically significant changes in mean haematology parameters were not adverse or not related to exposure to the test substance as the change did not occur in a dose-related pattern:
• Platelet count (PLT) was minimally increased in male rats dosed with 300 mg/kg/day (153% of control).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean clinical chemistry parameters at test day 29 in male or female rats. The following statistically significant changes in mean clinical chemistry parameters were considered to be potentially treatment related but non-adverse:

• Alanine aminotransferase (ALT) was minimally decreased in male and female rats at 100, 300, or 1000 mg/kg/day (variable statistical significance). These changes were minimal, were not clearly dose related, and there were no correlative changes in other associated liver enzyme parameters (AST, SDH, BILI) or liver histology. Therefore, the decreases in ALT in both male and female rats were considered to be potentially treatment related but non-adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean urinalyses parameters at test day 29 in male or female rats. The following statistically significant change in a mean urinalyses parameter was considered treatment related but non-adverse:

• pH was minimally decreased in female rats dosed with 1000 mg/kg/day (92% of control). Although individual urine pH values of only three female rats in the 1000 mg/kg/day group were lower than the lowest value observed in control rats (pH 6.0), they were not considered to be outside the range that is normally observed in female rats of this age and strain (95% CI for female rat urine pH 6.0 - 7.5). Therefore, this minimal change was considered to be treatment related but non-adverse.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on organ weights. The following statistically significant changes in the organ weight were considered non-adverse or unrelated to exposure to compound.

In males, mean absolute and mean relative (% brain weight) adrenal weights were increased 122% and 121% in the 1000 mg/kg/day exposure group, respectively, as compared to control values. Because these adrenal weight changes were not associated with changes in adrenal weight relative to body weight or with correlative microscopic findings, and because no adrenal weight changes occurred in female rats at any dose, they were considered spurious and unrelated to test substance administration.

In females, mean relative (% body weight) liver weight was increased 111% in the 1000 mg/kg/day exposure group as compared to the control value. Because the liver weight change was not associated with changes in absolute liver weight or relative to brain weight or with correlative microscopic findings, and because no liver weight changes occurred in male rats at any dose, this minor change in mean relative liver weight was considered spurious and unrelated to test substance administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross observations at the terminal necropsy were consistent with normal background lesions in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test substance-related microscopic findings. Microscopic changes likely occurring secondary to the wrapping procedure were present in the liver of a few rats across all the groups.

In a few animals across all the groups, including controls, there were microscopic observations in the liver, which were diagnosed as mild to moderate focally extensive necrosis. The lesion was characterized by focally extensive to multifocal areas of coagulative necrosis variably admixed with chronic inflammation. The lesion was primarily observed beneath the capsular surface but was also noted to extend into the hepatic parenchyma. The lesion was usually limited to one lobe only. In one animal, which died on test day 9, bridging necrosis accompanied with mixed inflammatory cell infiltration was noted.

These liver changes are similar to those previously reported to occur in rats on dermal studies. In those studies, the subcapsular liver changes were attributed to pressure on the liver by the abdominal wrap. Therefore, based on the morphology of the liver lesions observed in the current study, and their occurrence in both treated and control animals, these changes were considered to be secondary to the wrapping procedure and not primary test substance-related effects.

All other microscopic findings were consistent with normal background lesions in rats of this age and strain.
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant or treatment-related changes in coagulation parameters at test day 29 in male or female rats.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest dose tested
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.
Executive summary:

The objective of this study was to determine the potential toxicity of the test substance following repeated dermal exposure. The test substance was applied to the shaved, intact skin of male and female Crl:CD(SD) rats for 6 hours/day for 29 consecutive days. Three groups of 10 male and 10 female rats were treated dermally with 100, 300, or 1000 mg/kg/day of the test substance. A control group of 10 male and 10 female rats was similarly treated with deionized water. Body weights, food consumption, and detailed clinical observations were evaluated weekly. The animals were examined for acute clinical signs of systemic toxicity on days detailed clinical observations were not conducted. Ophthalmological assessments were performed prior to the start of exposure and near the end of the exposure period. Clinical and anatomic pathology endpoints were evaluated at the end of the exposure period.

 

No test substance-related deaths occurred. The deaths of one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group were attributed to their wrappings being too tight. No clinical signs of systemic toxicity were observed. No adverse or test substance-related effects occurred on body weights, body weight gains, food consumption, or food efficiency for any male or female dose group. No ophthalmological lesions were observed.

 

There were no test substance-related adverse effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology.

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 58 days (males); approximately 59 days (females)
- Weight at study initiation: 255.1-294.7 g (males); 174.9-221.1 g (females)
- Housing: Animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Enrichment (nylabone toy) was placed in each cage. Each cage rack contained only animals of one sex.
.- Diet (e.g. ad libitum): ad libitum, except when rats were fasted
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
The test material was moistened with water to form a thick paste
Details on exposure:
Approximately 24 hours prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. The target area to be treated (5 cm x 7.4 cm = 37 cm2) was marked on the back of each rat with a water-insoluble marker. The 37 cm2 treatment area is approximately equal to 10% of the total body surface area for rats in the 200 to 300 g range of body weights.

The test substance was moistened with deionized water to form a thick paste and applied in a thin and uniform layer to cover as much of surface of the target area as possible. The test site was covered with a 2-ply porous gauze pad followed by successive layers of stretch gauze (no more than 8 layers) and self-adhesive bandage. The control rats were treated with deionized water at the same volume as the high-dose rats. Some rats were fitted with plastic collars during the exposure period to prevent disruption of the wrappings. The rats were checked for dislodged wrappings several times daily during the exposure period.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 0, 100, 300, and 1000 mg/kg/day were selected for this study. The 1000 mg/kg/day dosage is a limit dose level. The other dosages were selected to establish a no-observed-adverse-effect level (NOAEL) and to assess a dose response for any observed effects.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least twice daily throughout the study. Abnormal behavior/appearance was recorded by exception.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before dosing on test day 0 and then weekly thereafter soon after test substance removal, each rat was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed on test day 0 and at weekly intervals during the study. All rats were weighed on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; The amount of food consumed by each rat was determined weekly by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. Food spillage < 5 g was not included in the calculation. From these measurements, mean daily food consumption over the interval was determined.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes; calculated from food consumption and body weight data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes; Two ophthalmology examinations were conducted by a veterinary ophthalmologist. The pretest examination was performed on all rats received for the study, prior to assignment to groups. Two rats with preexisting ophthalmology abnormalities were eliminated from consideration for use in the study. Surviving rats were examined prior to the final sacrifice. Both eyes of each rat were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes; carbon dioxide
- Animals fasted: Yes
- How many animals: All
- Parameters examined: red blood cell count, red cell distribution width, hemoglobin, absolute reticulocyte count, hematocrit, platelet count, mean corpuscular (cell) volume, white blood cell count, mean corpuscular (cell) hemoglobin, differential white blood cell count, mean corpuscular (cell) hemoglobin concentration, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes
- How many animals: All
- Parameters examined: aspartate aminotransferase, glucose, alanine aminotransferase, total protein, sorbitol dehydrogenase albumin, alkaline phosphatase, globulin, total bilirubin, calcium, urea nitrogen, inorganic phosphorus, creatinine, sodium, cholesterol, potassium, triglycerides, and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: quality, ketone, color, bilirubin, clarity, blood, volume, urobilinogen, specific gravity (SG), protein, pH, microscopic urine sediment examination, and glucose.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following tissues were collected from all of the rats:
Cardiovascular System: Heart and aorta
Digestive System: Liver, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum, salivary glands, and pancreas
Endocrine System: Pituitary gland, thyroid gland, parathyroid glands, and adrenal glands
Female Reproductive System: Ovaries (with oviducts), uterus (including cervix), mammary glands, and vagina
Hematopoietic System; Spleen, thymus, mandibular lymph node, mesenteric lymph node, bone marrow, and Peyer’s patches
Male Reproductive System: Testes, epididymides, prostate, and seminal vesicles with coagulating glands
Miscellaneous: Skin (treated and adjacent material), skin adjacent to the mammary gland, eyes (including retina and optic nerves), and gross observations
Musculoskeletal System: Skeletal muscle, femur/knee joint, and sternum
Nervous System: Brain (including cerebrum, midbrain, cerebellum, and medulla/pons), spinal cord (cervical, mid-thoracic, and lumbar sections), and sciatic nerve
Respiratory System: Lungs, trachea, nose (4 sections), larynx, and pharynx
Urinary System: Kidneys and urinary bladder

The following tissues were weighed from rats sacrificed by design at the end of the study: liver, kidneys, heart, spleen, thymus, adrenal glands, brain, testes, epididymides, accessory sex organs (prostate + seminal vesicles with coagulating glands), ovaries (with oviducts) and uterus (including cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated. Testes, epididymides, and eyes were fixed in modified Davidson’s solution. All other tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist. All collected tissues from rats in the control and 1000 mg/kg/day groups, and tissues collected from early decedents were processed to slides and evaluated microscopically.
Statistics:
For Body Weight, Body Weight Gain, Food Consumption, Food Efficiency, Clinical pathology,and Organ weights, use Levene’s test for homogeneity and the Shapiro-Wilk test for normality; If the preliminary test is not significant, use the One-way analysis of variance test followed by Dunnett’s test; If the preliminary test is significant, use the Kruskal-Wallis test followed by Dunn’s test. Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of systemic toxicity were observed. Incidental incidences of ocular or nasal discharge, scabs, wounds, wet fur, paleness, and stained skin/fur were not considered to be test substance related as the clinical signs were observed in both treated and control animals and/or the incidence did not occur in a dose-related pattern. Swollen head or nose observed in several animals was considered to be due to the wrapping procedure.
Dermal irritation:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the course of the study, one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group died on test days 14, 9, 9, and 13, respectively. The deaths of these rats were considered to be a result of the wrappings being too tight.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effect was observed on mean body weight in any male or female group. Mean body weights on test day 28 in the 1000 mg/kg/day groups were 101% and 98% of control (neither statistically significant) for males and females, respectively.

No adverse or test substance-related effect was observed on mean body weight gain in any male or female group. Mean overall (test days 0-28) body weight gain in the 1000 mg/kg/day male group was 101% of control (not statistically significant). Mean overall body weight gain in the 1000 mg/kg/day female group was 80% of control. This decrease in body weight gain was considered to be non-adverse because the decrease was not statistically significant compared to control and was driven by the low body weight gain of one animal.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related effect was observed on mean overall food consumption (test days 0-28) in any male or female group. The mean overall food consumption in the 1000 mg/kg/day groups was 102% and 99% of control (neither statistically significant) for males and females, respectively.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse or test substance-related effect was observed on mean overall food efficiency (test days 0-28) in any male or female group. The mean overall food efficiency in the 1000 mg/kg/day male group was 99% of control (not statistically significant). The mean overall food efficiency in the 1000 mg/kg/day female group was 83% of control (not statistically significant). This decrease correlated with the decreased body weight gain in that group and was considered to be non-adverse.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological changes were noted in male or female rats at any dose level.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or adverse changes in group mean haematology parameters at test day 29 in male or female rats. The following statistically significant changes in mean haematology parameters were not adverse or not related to exposure to the test substance as the change did not occur in a dose-related pattern:
• Platelet count (PLT) was minimally increased in male rats dosed with 300 mg/kg/day (153% of control).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean clinical chemistry parameters at test day 29 in male or female rats. The following statistically significant changes in mean clinical chemistry parameters were considered to be potentially treatment related but non-adverse:

• Alanine aminotransferase (ALT) was minimally decreased in male and female rats at 100, 300, or 1000 mg/kg/day (variable statistical significance). These changes were minimal, were not clearly dose related, and there were no correlative changes in other associated liver enzyme parameters (AST, SDH, BILI) or liver histology. Therefore, the decreases in ALT in both male and female rats were considered to be potentially treatment related but non-adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean urinalyses parameters at test day 29 in male or female rats. The following statistically significant change in a mean urinalyses parameter was considered treatment related but non-adverse:

• pH was minimally decreased in female rats dosed with 1000 mg/kg/day (92% of control). Although individual urine pH values of only three female rats in the 1000 mg/kg/day group were lower than the lowest value observed in control rats (pH 6.0), they were not considered to be outside the range that is normally observed in female rats of this age and strain (95% CI for female rat urine pH 6.0 - 7.5). Therefore, this minimal change was considered to be treatment related but non-adverse.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on organ weights. The following statistically significant changes in the organ weight were considered non-adverse or unrelated to exposure to compound.

In males, mean absolute and mean relative (% brain weight) adrenal weights were increased 122% and 121% in the 1000 mg/kg/day exposure group, respectively, as compared to control values. Because these adrenal weight changes were not associated with changes in adrenal weight relative to body weight or with correlative microscopic findings, and because no adrenal weight changes occurred in female rats at any dose, they were considered spurious and unrelated to test substance administration.

In females, mean relative (% body weight) liver weight was increased 111% in the 1000 mg/kg/day exposure group as compared to the control value. Because the liver weight change was not associated with changes in absolute liver weight or relative to brain weight or with correlative microscopic findings, and because no liver weight changes occurred in male rats at any dose, this minor change in mean relative liver weight was considered spurious and unrelated to test substance administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross observations at the terminal necropsy were consistent with normal background lesions in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test substance-related microscopic findings. Microscopic changes likely occurring secondary to the wrapping procedure were present in the liver of a few rats across all the groups.

In a few animals across all the groups, including controls, there were microscopic observations in the liver, which were diagnosed as mild to moderate focally extensive necrosis. The lesion was characterized by focally extensive to multifocal areas of coagulative necrosis variably admixed with chronic inflammation. The lesion was primarily observed beneath the capsular surface but was also noted to extend into the hepatic parenchyma. The lesion was usually limited to one lobe only. In one animal, which died on test day 9, bridging necrosis accompanied with mixed inflammatory cell infiltration was noted.

These liver changes are similar to those previously reported to occur in rats on dermal studies. In those studies, the subcapsular liver changes were attributed to pressure on the liver by the abdominal wrap. Therefore, based on the morphology of the liver lesions observed in the current study, and their occurrence in both treated and control animals, these changes were considered to be secondary to the wrapping procedure and not primary test substance-related effects.

All other microscopic findings were consistent with normal background lesions in rats of this age and strain.
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant or treatment-related changes in coagulation parameters at test day 29 in male or female rats.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest dose tested
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.
Executive summary:

The objective of this study was to determine the potential toxicity of the test substance following repeated dermal exposure. The test substance was applied to the shaved, intact skin of male and female Crl:CD(SD) rats for 6 hours/day for 29 consecutive days. Three groups of 10 male and 10 female rats were treated dermally with 100, 300, or 1000 mg/kg/day of the test substance. A control group of 10 male and 10 female rats was similarly treated with deionized water. Body weights, food consumption, and detailed clinical observations were evaluated weekly. The animals were examined for acute clinical signs of systemic toxicity on days detailed clinical observations were not conducted. Ophthalmological assessments were performed prior to the start of exposure and near the end of the exposure period. Clinical and anatomic pathology endpoints were evaluated at the end of the exposure period.

 

No test substance-related deaths occurred. The deaths of one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group were attributed to their wrappings being too tight. No clinical signs of systemic toxicity were observed. No adverse or test substance-related effects occurred on body weights, body weight gains, food consumption, or food efficiency for any male or female dose group. No ophthalmological lesions were observed.

 

There were no test substance-related adverse effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology.

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Additional information

Groups of 12 male and 12 female Alpk:APfSD (Wistar derived) rats were fed diets containing 0 (control), 100, 500 or 1250 ppm test substance for 90 consecutive days (equivalent to 0, 8.5, 41.7 or 104.9 mg/kg bw/day in males and 0, 9.7, 48.1 or 120.1 mg/kg bw/day in females). Clinical observations, bodyweights and food consumption were measured; and at the end of the scheduled period, the animals were killed and subjected to a full post mortem examination. Cardiac blood samples were taken; and urine samples were collected for clinical pathology, selected organs were weighed and specified tissues were taken for subsequent histopathology examination. One male given 1250 ppm test substance was terminated for humane reasons, unrelated to treatment with picoxystrobin, in week 7 of the study. All other animals survived to scheduled termination. There were no treatment-related clinical signs or ophthalmoscopy changes in any animals. High dose animals showed an initial decrease in bodyweight compared to concurrent controls, of approximately 7 and 4% in males and females respectively, during the first week of the study. At the end of the study, bodyweights in these animals were 10 and 8% below concurrent controls in males and females, respectively. Bodyweights in all other treated groups were not different from control values throughout the study. Food consumption was decreased throughout the study in 1250 ppm animals when compared to concurrent controls. Food consumption in other dosed groups was essentially similar to control values. Food utilisation was less efficient in high dose females during the first 4 weeks of the study but was similar to control values thereafter. Food utilisation efficiency in mid and low dose females and in all treated males was similar to control values. Reduced body weight is associated with decreased food consumption. Decreased food consumption (e.g. as a result of changed taste) in young rats is known to have long-lasting adverse effects on growth and development. Reduced body weight is not adverse as evidenced by decreased mortality and age related morbidity in rats fed high doses compared to controls. Minor statistically significant changes in some red cell parameters were seen in the 1250 ppm animals. Given the small magnitude (1.5-3% difference from control) and the absence of changes in other corresponding haematology parameters, these minor changes were considered to be of no toxicological significance. Slight decreases in plasma triglycerides in high dose males, increases in bilirubin in high dose females and increases in cholesterol in high and mid dose females were seen. In the absence of any histopathological change in the liver these differences were considered to be of no toxicological significance.Statistically lower ALT & AST observed in this study were only in high dose males. The decreases are less than 50% compared to control and considered small. While large increases (>50%) may be associated with adverse effects, small decreases are generally regarded as “not adverse” because they cannot be correlated with toxicologically relevant findings (Hall et al., 2012, Liver Hypertrophy: A review of adaptive changes – Conclusions from 3rd International ESTP Expert Workshop, Toxicologic Pathology 40:971-994). Consistent with this conclusion, the differences in this study were not associated with target organ toxicity. Decreases in ALP were also observed at 500 (males only) & 1250 ppm (males and females.) Based on the consistency of the finding, it is likely test substance related. However, based on the direction of change and lack of associated target organ toxicity, they are considered non-adverse.The changes in ALT, AST, & ALP were not reproducible in the more recent chronic study where rats were fed up to 3500 ppm. The reduction of enzymes activity is clearly treatment-related and may be linked to the fungicide mode of action (block mitochondrial electron transport at the Qo site of complex III, reducing ATP production and inhibiting cellular respiration); it could therefore be of toxicological relevance.The same MoA was observed for azoxystrobin or other strobilurin fungicides. German authorities confirmed with human medical data that reduction of these enzymes activity does actually not represent adverse effects. Therefore, the only potentially adverse effect observed at 500 ppm is an increase in bilirubin and haematological changes.There was an increase in liver weight, after adjustment for terminal bodyweight, in both sexes at the high dose level and in males at 500 ppm. In the absence of any histopathological change in the liver, this was considered to be a result of non-adverse induction of hepatic metabolising enzymes and an adaptive response to exposure of a xenobiotic. There were no treatment-related gross pathological or histopathological changes in any organs. The NOAEL was 500 ppm (41.7 mg/kg bw/day in males and 48.1 mg/kg bw/day in females) based on reduced body weight and food consumption at the highest dose of 1250 ppm.

 

Groups of ten male and ten female C57BL/10JfAP/Alpk mice were fed diets containing 0 (control), 200, 800, 1600 or 2400 ppm of test substance for 90 consecutive days (equivalent to 0, 33.2, 137.3, 290.8 or 421.6 mg/kg bw/day in males and 0, 43.8, 176.1, 358.5 or 534.8 mg/kg bw/day in females). Clinical observations, bodyweights and food consumption were measured and at the end of the scheduled period, the animals were sacrificed and subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. There were no clinical changes considered to be related to treatment with the test substance. There were reductions in food consumption and bodyweight, compared to concurrent control animals, and increases in liver weight, after adjustment for terminal bodyweight, in animals at dose levels of 800, 1600 or 2400 ppm test substance. Food utilisation was less efficient in animals give 1600 or 2400 ppm. There was a treatment related increase in the incidence of minimal hepatocyte hypertrophy in the livers of males given 1600 or 2400 ppm and in livers of females given 800, 1600 or 2400 ppm. The changes in liver weight and accompanying hepatocellular hypertrophy are considered to be a result of non-adverse induction of hepatic metabolising enzyme and an adaptive response to exposure of a xenobiotic. The NOAEL was 200 ppm (equivalent to 33.2 and 43.8 mg/kg bw/day in males and females, respectively).

 

Groups of four male and four female beagle dogs were fed diets containing 0 (control), 125, 250 or 500 ppm test substance for a period of at least 90 days (equivalent to 0, 4.3, 8.9 or 16.5 mg/kg bw/day in males and 0, 4.3, 8.5 or 16.9 mg/kg bw/day in females). Clinical observations and veterinary examinations (including ophthalmoscopy) were made and bodyweights, food consumption and clinical pathology parameters were measured. At the end of the scheduled period, the animals were subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. Slightly increased incidences of fluid faeces in males and salivation in females were seen in animals given 500 ppm test substance. Both incidences were minimal and were concluded to be of no toxicological significance. There were no veterinary or ophthalmoscopic findings that were considered to be related to administration of the test substance. Treatment related effects on bodyweight were seen for males and females given 500 ppm. A minimal reduction (4%, week 14 of the study) in body weight was also seen in males receiving 250 ppm; however this degree of body weight change is not considered either biologically or toxicologically important. Food consumption was reduced in both sexes at 500 ppm. Throughout most weeks of the study, group mean food consumption for males and females receiving 500 ppm was slightly reduced, in comparison with that of concurrent controls, with these differences usually attaining statistical significance. For animals receiving 125 or 250 ppm, there were no treatment-related effects on food consumption for the duration of the study. Group mean platelet counts, adjusted for pre-experimental values, were slightly raised throughout the study for males receiving 500 ppm and during weeks 4 and 8 for males receiving 250 ppm. The statistically significant increases in mean platelet values in male dogs fed 250 and 500 ppm are not considered to be related to treatment, but consistent with normal biological variation based on the following: 1) the differences in platelet counts in individual treated dogs relative to their respective pretest values were similar to those of controls at all time points; 2) there was no evidence of any changes that would be expected to be associated with an increase in platelet counts, such as inflammation or haematological effects; 3) there were no statistically significant changes in platelet counts in female dogs in the study at any dose level; and 4) there were no statistically significant changes in platelet counts in male or female dogs in the one-year study where animals were fed diets up to the same high concentration of 500 ppm. There were no changes in any other haematology parameters measured that could be attributed to administration of the test substance. Minor decreases in group mean plasma albumin, and associated total protein, were seen in high dose animals. A statistically significant decrease in albumin was also noted at 250 ppm at week 4 only. These changes are not considered of toxicological relevance based on examination of plasma albumin data on an individual animal basis that revealed the following: 1) albumin levels of both control and all treated dogs varied less than 10% of pretest values during the course of the study; 2) pretest values for the 250 and 500 ppm groups were lower than the controls, thereby making statistical comparisons of group means in this case of limited value; 3) values for controls and the 125 and 250 ppm treated groups varied during the course of the study with values generally ending either equal to pretest or slightly higher; 4) at 500 ppm the reduced albumin levels at week 4 may represent a minimal effect of treatment, likely secondary to reduced body weight and food consumption; however, these changes are minimal and not progressive in severity with increased duration of dosing as values at study end were equal or greater than at week 4; 5) there are no study findings suggestive of an adverse dysproteinemia, such as protein-losing enteropathy or nephropathy, or hepatic insufficiency; and 6) there were no statistically significant changes in albumin in male or female dogs in the one-year study where animals were fed diets up to the same high concentration of 500 ppm. Therefore, the changes in plasma proteins (lower albumin and an associated lower total protein) are not considered adverse due to their minimal nature and lack of associated adverse changes known to produce hypoproteinemia. There were no changes in any other blood clinical chemistry parameters measured that were attributed to administration of the test substance. Occasional changes regarding platelet count and albumin production were observed at 250 ppm, and consistently observed at the high dose, where according to the experts (EFSA Pesticides Peer Review Meeting, 23-26 February 2016) they were considered adverse, together with reduced body weight, food consumption and clinical signs. Group mean left, right and combined kidney weights, adjusted for terminal bodyweight, for males receiving 500 ppm were marginally higher (combined weights were approximately 12% higher) than that of concurrent controls. However, these differences only attained statistical significance for the right kidney weight. As this effect was not associated with any treatment-related adverse histopathological changes in the kidney of males it was considered to be of no toxicological significance. Minimal focal unilateral tubular dilatation with eosinophilic casts was observed in the kidneys of 3/4 females receiving 500 ppm test substance. This was also seen in the kidneys in 1/4 males in the 250 ppm group and in 1/4 males at 500 ppm. However, single incidences of this finding had been seen historically in both sexes on 90-day studies in the testing laboratory, and therefore, in the absence of a dose-related increase, this was not attributed to administration of the test substance. The NOAEL was 250 ppm in males and females (equivalent to 8.9 and 8.5 mg/kg bw/day, respectively).

 

Groups of four male and four female beagle dogs were fed diets containing 0 (control), 50, 150 or 500 ppm test substance for a period of at least 1 year (equivalent to 0, 1.59, 4.80 or 16.08 mg/kg bw/day in males and 0, 1.56, 4.57 or 15.71 mg/kg bw/day in females). Clinical observations and veterinary examinations (including ophthalmoscopy) were made and bodyweights, food consumption and clinical pathology parameters were measured periodically throughout the study. At the end of the scheduled period, the animals were subjected to a full examination post mortem. Selected organs were weighed and specified tissues were taken for subsequent histopathology examination. None of the animals died before the scheduled termination. Administration of 500 ppm test substance to female dogs resulted in an increased incidence of the observation of “thin appearance” which is related to effects on bodyweight at this dose level. There was an increased incidence of “reddened gums” and of fluid faeces in males receiving 500 ppm. However, in view of the low overall incidences of these changes and the lack of association with any histopathological change, the differences from control were considered to be of no toxicological significance. There were no veterinary or ophthalmoscopy findings that were considered to be related to administration of the test substance. Dietary administration of 500 ppm to male and female dogs resulted in reduced bodyweight, with a maximal effect in males of 11% at week 26 and in females 15% at week 36. There were no effects on bodyweight at 50 or 150 ppm. Reduced food consumption was seen in both sexes at 500 ppm. There were no treatment-related effects at 50 or 150 ppm. There were no treatment-related effects on haematology, clinical chemistry and urinalysis parameters. At necropsy, group mean thyroid weights, adjusted for terminal bodyweight were higher than concurrent controls for females receiving 500 ppm (0.74 g) test substance. However, in the absence of any associated treatment-related histopathological changes and bearing in mind that the control group mean value was low (0.50 g) compared with the historical control group mean range (0.65-0.94 g), the increase in thyroid weight was considered to be of no toxicological significance. There were no macroscopic or microscopic pathology findings attributed to administration of the test substance. The NOAEL was 150 ppm (equivalent to 4.8 mg/kg bw/day in males and 4.6 mg/kg bw/day in females).

 

Five groups of 80 rats/sex/group received untreated diet or the test article orally via dietary admixture approximately 105 weeks (males) or 103 weeks (females) at diet concentrations of 0, 50, 200, 1000, and 3500 ppm. Following 12 months of treatment, ten animals/sex/group were submitted to an interim necropsy. Observations for morbidity, mortality, injury, body weights, body weight gain, food consumption, food efficiency, and compound consumption were reported. Ophthalmoscopic examinations, clinical pathology, organ weights, and gross and histopathologic examinations were performed. Survival in the 3500 ppm male and female groups and in the 1000 ppm female group was significantly greater than in controls. This increase is likely due to lower body weight in these groups (not statistically significant in females at 1000 ppm). There were no adverse clinical or ophthalmological observations attributed to test article exposure. There was an increase in the incidence of soft faeces at 1000 and 3500 ppm primarily in males that was considered possibly test article-related but not adverse. Mean body weight and body weight gain were reduced during the study in both sexes at 3500 ppm. All of these differences were statistically significant except the male final body weight and overall body weight gain. However, these values were significantly different from control for most of the study. These body weight findings were associated with significantly lower mean food consumption and food efficiency over the first year at this exposure level which continued for the duration of the study (variable statistical significance). Body weight and nutritional parameters in lower concentration groups were generally comparable to control over the study.  No test article-related effects were noted on any clinical pathology parameters, organ weights, macroscopic findings, or incidence of masses. There were no test article-related microscopic findings following 1 year of treatment. At the end of the study, statistically significant increases in the incidences of interstitial cell hyperplasia and benign adenoma in the testes were observed in male rats at 3500 ppm. Although survival was increased, the majority of adenomas and hyperplasia occurred in terminal or near terminal animals, and the percent incidence of adenomas fell within the historical limits of the laboratory, it is considered likely that the increases in testicular interstitial cell adenoma and hyperplasia in the 3500 ppm males were related to exposure to the test article. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 1000 ppm, equivalent to 45.3 and 57.1 mg/kg/day in males and females, respectively. This NOAEL is based on reduced body weight and nutritional parameters observed in both sexes at 3500 ppm and increased incidence of interstitial cell hyperplasia and benign adenoma in the testes in males at 3500 ppm (equivalent to 162.1 and 203.3 mg/kg/day for males and females, respectively).

 

Five groups of young adult male and female Crlj:CD1(ICR) mice (60/sex/group) were administered diets that contained 0, 100, 600, 2400, or 4800 ppm of the test substance for approximately 18 months. Body weights and food consumption were evaluated and ophthalmological assessments were performed. White blood cell differential counts were evaluated in surviving mice at the end of the exposure period and in mice that were sacrificed in extremis. After approximately 18 months of dietary exposure, mice were sacrificed and given a gross and microscopic pathological examination. No test article-related changes were observed in the following observations in male and female mice fed up to 4800 ppm of the test substance: clinical observation, body weight parameters, food intake parameters, ophthalmology, white blood cell differential counts, cause of death, and gross pathological parameters, and neoplastic changes. Test article-related and biologically adverse microscopic findings were limited to non-neoplastic effects in the duodenum and consisted of increased incidences and severity of mucosal hyperplasia in males fed dietary concentrations of 2400 or 4800 ppm. Female mice did not exhibit treatment-related duodenal mucosal hyperplasia at any concentration. Test article-related (non-adverse) increases in liver weights were observed in males and females fed dietary concentrations of ≥2400 ppm. In female mice, the increased liver weights correlated with the test article-related microscopic finding of hepatocellular hypertrophy. Both the liver weight increases and hepatocellular hypertrophy were consistent with hepatic enzyme induction and were interpreted to be not adverse. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 600 ppm for male mice and 4800 ppm for female mice, equivalent to 71 and 799 mg/kg/day, respectively.

 

Groups of 5 male and 5 female rats were administered the test substance at 0, 200, 500 or 1000 mg/kg bw per day by dermal application 5 days/week over a 28-day period (20 applications).  The test material was moistened with water and applied daily to clipped skin, which was then covered with an occlusive dressing for 6 hours. Clinical observations, body weight and feed consumption were recorded throughout the study. At the end of the scheduled period, the animals were killed and subjected to a post-mortem examination. Cardiac blood samples were taken for clinical pathology (haematology and blood clinical chemistry), selected organs were weighed and tissues were taken for histopathological examination. Other than an apparent increase in platelets that was linked to a low control value, there were no adverse systemic effects. An increase in sloughing at the application site was noted in high dose males. The NOAEL was 1000 mg/kg bw/day, the highest dose tested.

 

The test substance, suspended in air, was administered to rats via nose-only inhalation for 6 hours per day, 5 days a week over a 4-week period for a total of 20 exposures. Four groups of five male and five female rats were exposed to 0 (air control), 0.001, 0.01, or 0.025 mg/L of the test substance. To determine reversibility of potential adverse effects, satellite groups of five male and five female rats were exposed to 0 (air control) or 0.010 mg/L test substance for a total of 20 exposures, and allowed to recover two weeks before sacrifice. Animals were observed for clinical signs of toxicity daily, had body weights measured twice weekly, and food/water consumption parameters determined daily, but reported weekly. Three to four days following the final exposure, groups of 10 (control, low, intermediate, and high concentration) male and female rats were fasted overnight, blood and urine samples were collected for clinical pathology evaluations, and the rats were sacrificed for anatomic pathology evaluation, including microscopic tissue evaluation. Following a 2-week recovery period, the remaining 10 male and 10 female rats from the satellite control and intermediate exposure groups were fasted overnight; blood and urine samples were collected, and the animals were sacrificed for evaluation of anatomic pathology endpoints. Exposure to the test substance did not result in test substance related adverse changes in body weights, weekly feed and water consumption, and no clinical signs of toxicity were observed over the course of the study. There were no adverse changes in clinical pathology parameters. There were no test substance-related organ weight changes or gross pathological observations. No test substance related changes in microscopic histopathology were observed. A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration, but this was not considered substance related. The no-observed-adverse-effect concentration (NOAEC) was 0.025 mg/Lfor male and female rats, the highest concentration tested. 

Justification for classification or non-classification

Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health. There is no evidence for any adverse findings or serious target organ toxicity in 90-day repeat dosing studies in rats, mice or dogs and in a 1-year feeding study in dogs that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. The primary effects in these feeding studies were reductions in body weight and food consumption. Increases in liver weight in rats and mice and hepatocellular hypertrophy in mice were observed at high doses. In the absence of liver cell toxicity, these changes are attributed to no adverse induction of hepatic metabolising enzymes, an adaptive response to xenobiotic exposure. The test substance did not induce any adverse effects on the nervous system or immune system, nor when tested by the dermal and inhalation routes in short-term repeat exposure studies. An increased incidence and severity of mucosal hyperplasia was observed in male mice fed diets containing test substance at 2400 ppm (293 mg/kg bw/day) and 4800 ppm (583 mg/kg bw/day) for 18 months. However, the dose levels at which this finding were observed are considerably greater than the STOT RE guidance value for Category 2 (10-100 mg/kg bw for the oral route) when extrapolated to a 90-day study using Haber’s rule (1758 mg/kg bw/day). Therefore, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.