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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 July to 1 October 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Since the study was considered a range-finding study to select dose levels for a 2-year carcinogenicity study in rats, the potential deviations from OECD 408 did not impact the intended purpose of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Powder
Details on test material:
- Description: Solid (Powder)

Test animals

Species:
rat
Strain:
other: Crl:CD® (SD)IGS BR
Details on species / strain selection:
The rat is an animal model commonly utilized in toxicity studies. In addition, a historical data base is available for comparative evaluation.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Kingston, New York 12484.
- Females were nulliparous and non-pregnant: Yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: Male: 226 g (202-245 g); Female: 167 g (147-186 g); Individual weights of animals placed on test were within ±20% of the mean weight for each sex.
- Housing: Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter.
- Diet: Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St.Louis, Missouri) was available ad libitum. Fresh food was presented twice weekly.
- Water: Water was available without restriction via an automated watering system (Elizabethtown Water Company, Westfield, New Jersey).
- Acclimation period: 17 days

DETAILS OF FOOD AND WATER QUALITY
There were no known contaminants in the feed or water which were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 15-24 °C
- Humidity: 38-88 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 1 July to 1 October 1999

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
other: 0.2% Ascorbic Acid in Propylene Glycol
Details on oral exposure:
TEST ITEM STOCK SOLUTION
In a nitrogen-filled glove bag, an appropriate amount of test item was weighed onto a weigh boat/paper or directly into a calibrated beaker. An additional 10% of the test item was added to the Group 5 stock solution only, in order to achieve targeted concentrations after four days of feed presentation. This was determined based on pretest stability analyses. At each weighing interval, a new package of test item was used. The total final volume was achieved by adding an appropriate amount of control item solution. The mixture was stirred for at least one hour or until a clear solution was obtained. The stock solution was nitrogen-capped after each use.

PREPARATION OF DIETS
Group 1: Inside a nitrogen-filled glove bag, the appropriate amount of control item solution for each sex was measured into a transfer container using a syringe.
Groups 2-5: Inside nitrogen-filled glove bags, appropriate amounts of stock solution and control item solution for each group/sex were measured into transfer containers using syringes. The transfer containers were capped tightly and remained nitrogen-capped until just prior to adding to the diet.
Appropriately identified dose group buckets were used to weigh and store the test diets. The appropriate amount of Certified Rodent Diet No. 5002 was weighed into each bucket. For each dose group/sex, approximately 100 grams of untreated diet from the bucket was added to a mortar. The control item solution or test item solution was poured from the transfer container into the mortar. Additional untreated diet from the bucket was added as needed and mixed in the mortar with a pestle until homogenous. The transfer container was rinsed with several grams of untreated diet from the bucket. The rinse was added to the contents of the mortar and mixed with a pestle until homogenous. Approximately half of the untreated diet from the bucket was placed into the bowl of a mixer. The contents of the mortar was poured onto the layer of diet in the bowl of the mixer. The mortar and pestle were rinsed with several grams of untreated diet from the bucket and this rinse was added to the bowl of the mixer. The remaining untreated diet in the bucket was added to the mixer. Using a paddle blade, the untreated diet and mortar contents were mixed for approximately 15 minutes.

CONTROL ITEM SOLUTION: 0.2% Ascorbic Acid in Propylene Glycol
The appropriate amount of ascorbic acid was weighed onto a weigh boat/paper, or directly into a calibrated beaker. A 2% solution was achieved by adding an appropriate volume of Propylene Glycol. The mixture was stirred overnight, using a stirbar and stir plate, until a clear solution was obtained. The mixture was protected from light and nitrogen-capped while stirring. The control item solution was prepared weekly and stored refrigerated, protected from light and nitrogen capped.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF TEST DIETS: Analyses to determine homogeneity, stability, and concentration of the test and/or control diets with carriers under the conditions of this study were performed by the Testing Facility.
HOMOGENEITY: Prior to initiation of the study, batches of low-concentration and high concentration diets were prepared. Three samples each from the top, middle and bottom portion of each mixture were taken for analysis.
STABILITY: Duplicate samples of the low-, high-mid-, and high-concentration diets were assayed 1, 2, 3 and 4 days after preparation.
CONFIRMATION ANALYSIS: All four dietary levels were assayed at Weeks 1, 2, 3, 4, 8 and 13 (2 samples per concentration).
Duration of treatment / exposure:
3 months
Frequency of treatment:
The test item was administered continuously in the diet. Each animal received the appropriate amount of the test item in the diet, ad libitum, 7 days per week, for at least 3 months. Test item administration continued through the day prior to necropsy. Animals were fed twice weekly (4- and 3-day feed presentation).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 4
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Group 5
No. of animals per sex per dose:
10
Control animals:
other: Control animals received untreated diet, and the same volume of 0.2% ascorbic acid and propylene glycol that was used as the premix for the high-dose group.
Details on study design:
- Dose selection rationale: Doses were selected by the Sponsor based on multiples of the predicted oral ingestion of the test item.
- Rationale for animal assignment: More animals than required for the study were purchased and acclimated. Animals considered unsuitable for the study on the basis of pretest physical examinations, outlying body weight data, or ophthalmoscopic examinations evaluations were eliminated prior to random selection for group assignment. Animals considered suitable for study were randomly assigned into 5 groups of 10 animal per sex by a computerized random sort program so that body weight means for each group were comparable.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations were made once daily for any signs of poor health or toxic or pharmacologic effects (e.g., abnormalities in general condition, appearance, activity, behavior, respiration, etc.).
Animals were observed in their cages twice daily for mortality and signs of severe toxic or pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were removed from their cages and examined twice pretest and weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., selfmutilation, walking backward) were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were removed from their cages and weighed twice pretest, twice weekly during treatment and terminally (after fasting). Terminal, fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed was available without restriction 7 days/week. Animals were presented with full feeders weighing 570 ± 3 grams during the initial pretest feed consumption period (Day -6) and 500 ± 3 grams thereafter. Full feeder weights include the weight of the feed, jar and lid. After 3 or 4 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the 3- or 4-day period. Feed consumption was measured (weighed) biweekly, beginning one week prior to treatment.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Calculation: grams of food consumed/kilogram of body weight/day (g/kg bw/day) = grams of food consumed / body weight (kg)* / 3 or 4 days

TEST ITEM INTAKE
Calculated from food consumption data and based on nominal dietary concentrations.
Calculation: mg of test item consumed/kilogram of body weight/day (mg/kg bw/day) = food consumed (g/kg/day) x dietary concentration (mg test item/g of diet)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Lids, lacrimal apparatus and conjunctiva were examined visually for all animals (10/sex/group) pretest (Day -8) and at study termination (Day 91).
- The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. Mydriacyl 1 % was used to induce mydriasis.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood for haematology studies (approximately 0.5 mL) was collected from all animals (10/sex/group) at study termination into tubes containing EDTA anticoagulant. Blood for coagulation studies (approximately 1.0 mL) was collected from all animals (10/sex/group) at study termination into tubes containing sodium citrate anticoagulant.
Blood for clinical chemistry studies was collected from all animals (10/sex/group) at study termination into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum.
- Anaesthetic used for blood collection: Yes; Blood for haematology and coagulation studies were obtained via the orbital sinus (retrobulbar venous plexus) under light CO2/O2 anesthesia. Blood samples for clinical chemistry studies were obtained from the aorta at necropsy.
- Animals fasted: Yes; Animals were fasted overnight prior to blood collection.
Parameters
- Haematology: Hemoglobin concentration, Hematocrit, Erythrocyte count, Platelet count, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Total leukocyte counts, Differential leukocyte count, Absolute lymphocytes (calculated value; total WBC x % lymphocyte value /100), Absolute segmented neutrophils (calculated value; total WBC x % segmented neutrophil /100)
- Coagulation: Prothrombin time, Activated partial thromboplastin time
- Clinical chemistry: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Blood urea nitrogen, Creatinine, Glucose, Creatine kinase, Cholesterol- enzymatic, Triglycerides, Total protein, Albumin, Globulin, Albumin/globulin ratio, Total bilirubin, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma-glutamyl transferase, T3 and TSH

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Testing was staggered over four sessions with approximately equal numbers of animals per sex per dose group in each session.
Dose groups that were examined:
MOTOR ACTIVITY: Using a modified version of Schulze's procedures (Schulze, 1990), the locomotor activity of all animals (10/sex/group) was monitored pretest and at study termination using an automated Photobeam Activity System (San Diego Instruments, Inc., San Diego, California).
FUNCTIONAL OBSERVATIONAL BATTERY: A functional observational battery of behavioral tests (Moser, 1989) was performed on all animals (10/sex/group) pretest and at study termination. The following evaluations were performed as part of the functional observational battery: Home Cage Evaluations; Handling Evaluations; Open Field Evaluations; Reflex Assessments; Grip Strength; Landing Foot Splay; Air Righting Ability

IMMUNOLOGY: No
Sacrifice and pathology:
NECROPSY
- All animals (10/sex/group) were sacrificed at study termination (Test Days 92 and 93) by exsanguination following carbon dioxide/oxygen inhalation.
A necropsy schedule was established to ensure that approximately equal numbers of males and females were examined on each day of necropsy and that examination of animals of both sexes were performed at similar times of the day throughout the necropsy period. In addition, animals were sacrificed in the following order: one rat each was selected from the control, high-, high-mid-, low-mid-, and low-dose groups and sacrificed in that order. This sequence was repeated until all the rats were sacrificed.

GROSS PATHOLOGY: Yes; Complete macroscopic postmortem examinations were performed immediately after death on all animals killed study termination. The macroscopic postmortem examination included examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities. Animals were fasted prior to scheduled sacrifices.

ORGAN WEIGHTS
Organs indicated in Table 7.5.1/1 were weighed for all animals at study termination. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together/separately.

HISTOPATHOLOGY: Yes
Tissues preserved and examined histopathologically: The tissues listed in Table 7.5.1/1 were obtained at terminal sacrifice and preserved for all animals. In addition, slides of the indicated tissues were prepared and examined microscopically for all animals. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded.
Preservatives: All tissues - 10% neutral buffered formalin.
Eyes were placed in glutaraldehyde/paraformaldehyde initially and then retained in 10% formalin. Testes and epididymides were placed in Bouin's solution initially and then retained in 10% formalin. Lungs, and urinary bladder were infused with formalin prior to their immersion into a larger volume of the same fixative.
Processing: After fixation, the tissues and organs from all animals were routinely processed, embedded in paraffin, cut at a microtome setting of 4-7 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy. The bones were decalcified in formic acid.
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All physical observations considered common findings in laboratory rats and have been seen previously in this laboratory. No test item related effects were evident.
Mortality:
no mortality observed
Description (incidence):
All animals survived until study termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean body weights of males administered 200 and 400 mg/kg bw/day of test item were slightly lower (5-6%) relative to control values at termination of the study. There were no statistically significant differences between mean body weights or cumulative weight gains of control and treated males.
- Mean body weights of males administered 50 and 100 mg/kg bw/day and all treated female groups were considered comparable to their respective control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- No effects due to dietary administration of test item were evident in the feed consumption data.
- Mean test item intake values were 48.5, 97.0, 193.2 and 386.0 mg/kg bw/day (males) & 48.6, 97.8, 195.2 and 391.4 mg/kg bw/day (females) corresponding to 50, 100, 200 and 400 mg/kg bw/day, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmology examinations at termination revealed no effects attributed to administration of test item.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item related effects were seen in the hematology parameters examined at termination of the study.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related effects were seen in the clinical chemistry parameters examined at termination of the study.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
MOTOR ACTIVITY: No test item related changes were evident in motor activity for either sex in any of the treatment groups.
FUNCTIONAL OBSERVATIONAL BATTERY: Test item administration did not affect the neurological condition of the animals as measured by a functional observational battery of assessments. Landing foot splay and grip strength for all treated groups were comparable to control values or within the range of normal variation. No abnormal movements were observed in any of the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effect of test item administration was evident in the absolute or relative organ weights at study termination. Values of treated groups were similar to control values or exhibited normal variability.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings attributable to treatment with test item.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Thyroids were examined from controls and animals fed test item at 100,200 and 400 mg/kg bw/day. Parathyroids were examined from controls and animals fed test item at 400 mg/kg bw/day . The follicular epithelial cells of the thyroid showed a range of secretory activity in animals from all dose levels examined including the control group. This was not influenced by administration of test item.
- Macroscopic abnormalities were also examined microscopically. In most cases the abnormalities reflected the spontaneous lesions commonly seen in this species. However, in the case of one abnormality, (cortical cysts in the kidney: Animal No. 5060) the microscopic findings were not typical of a spontaneous lesion. Focal areas of marked tubular vacuolation were present. In some cases these had developed into large cysts (correlating with the macroscopic finding) but in other cases they were associated with a focal atypical hyperplasia comprising solid nests of vacuolated cells.
- On the basis of this finding, the kidneys from all animals in the control and 400 mg/kg bw/day group were examined. No further evidence of this lesion was seen.
- An unusual vacuolation of the tubular epithelial cells of the kidney, which was associated with cystic dilatation and an atypical hyperplasia, was seen in one female administered 400 mg/kg bw/day. Since no other animals in this dose group showed any evidence of this lesion, it is considered to be incidental and unrelated to treatment with test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 386 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 391.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

ANALYTICAL RESULTS:

Analysis of preliminary mixes confirmed that the preparation procedure used for this study produced homogeneous mixtures and that the test item was stable in the diet, under storage conditions used in this study, for at least four days. Analyses conducted during the treatment period confirmed that diets of appropriate concentration were administered. Mean analytical concentrations, expressed as percent of nominal (desired) concentrations were as follows:

 

GROUP

NOMINAL CONCENTRATION (mg/kg bw/day)

ANALYTICAL CONCENTRATION (% of Nominal)

Male

Female

2

50

100.2

103.2

3

100

99.5

100.0

4

200

92.1

95.5

5

400

98.1

97.8

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the systemic parental NOAEL was considered to be 400 mg/kg bw/day nominal in male and female rats (i.e. 386.0 mg kg bw actual dose in males and 391.4mg kg bw actual dose in females), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.
Executive summary:

In a repeated dose toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, the test item was administered orally, via dietary admixture, to Sprague-Dawley CD® rats (10/sex/group) at the dose levels of 50, 100, 200 and 400 mg/kg bw/day for a period of at least 3 months. Physical observations, ophthalmoscopic examinations, body weight, food consumption, motor activity and functional observational battery evaluations were performed on all animals pretest and at selected intervals during the treatment period. Hematology and clinical chemistry were performed on all animals at study termination. After at least three months of treatment, all animals were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were conducted on all animals. Histopathological evaluations were conducted on the kidneys from all animals in the control and high-dose groups and on the thyroid/parathyroid glands from all animals in the control, mid-, high-mid- and high-dose groups. Analyses conducted during the treatment period confirmed that diets of the appropriate concentrations were administered. Mean test item actual intake values were 48.5, 97.0, 193.2 and 386.0 mg/kg bw/day (males) & 48.6, 97.8, 195.2 and 391.4 mg/kg bw/day (females) corresponding to 50, 100, 200 and 400 mg/kg bw/day, respectively.

All physical observations considered common findings in laboratory rats and have been seen previously in this laboratory. No test item related effects were evident. All animals survived until study termination.

At 200 and 400 mg/kg bw/day, a slight decrease in body weight (5-6% relative to control weights) in males, however, differences were not statistically significant and were considered minimal. Body weights of female rats fed the same dietary levels were considered comparable to control weights. No effects due to dietary administration of test item were evident in the feed consumption data.

Ophthalmology examinations at termination revealed no effects attributed to administration of test item. No test item related effects were seen in the hematology parameters examined at termination of the study. No test item related effects were seen in the clinical chemistry parameters examined at termination of the study. No test item related changes were evident in motor activity or functional observational battery.

No effect of test item administration was evident in the absolute or relative organ weights at study termination. Values of treated groups were similar to control values or exhibited normal variability. There were no macroscopic findings attributable to treatment with test item. Microscopic examination of selected tissues (thyroids/parathyroids and kidneys) revealed no test item related effects. An unusual vacuolation of the tubular epithelial cells of the kidney, which was associated with cystic dilatation and an atypical hyperplasia, was seen in one female administered 400 mg/kg bw/day. Since no other animals in this dose group showed any evidence of this lesion, it is considered to be incidental and unrelated to treatment with test item.

Under the test conditions, the systemic parental NOAEL was considered to be 400 mg/kg bw/day nominal in male and female rats (i.e. 386.0 mg kg bw actual dose in males and 391.4mg kg bw actual dose in females), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.