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Description of key information

The NOAEL for repeat dose toxicity conducted by the oral route was considered to be 1000 mg/kg/day in both sexes under the conditions of this study, as there were no effects of parameters associated with repeated dose toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Lot no.: 140220
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Centre).
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 320.9-367.7 g; Females: 214.9-240.8 g
- Housing: Males and females (except during the gestation and lactation periods); stainless steel cages. For females during the gestation and lactation periods; polymethylpentane cages.
- Diet (e.g. ad libitum): Ad libitum (except during the urine collection and during measurement of motor activity) autoclaved sterilized pellet diet (CRF-1, oriental yeast co. Ltd.). Animals were fasted on the day before scheduled necropsy.
- Water (e.g. ad libitum): Ad libitum (except during the measurement of motor activity) well water.
- Acclimation period: 6 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24.3
- Humidity (%): 48.6 to 69.8
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 0.5% w/v% methyl cellulose aqueous solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing preparations were prepared once every 4-8 days (based on the results of previous stability and homogeniety assessments). The test substance was initially ground in an agate pestle and mortar, and a few drops of vehicle added. This suspension was then transferred into a measuring cylinder. The appropriate volumes of the vehicle was added to the measuring cyclinder and mixed to achieve the required concentrations which were then added into brown glass vials and stored refrigerated until use.

VEHICLE
- Concentration in vehicle: 0, 11, 33 and 100 mg/mL
- Amount of vehicle (if gavage): Dose volume of 10 mL/kg/day
- Lot/batch no. (if required): PDG1792
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the initial test substance preparation, 15 mL of each of the analytical samples were tested for homogeneity and analytical determination. The measured concentrations were within acceptable ranges (actual values 98.4 to 100.3% of nominal).
Duration of treatment / exposure:
Males: From 14 days before mating (Days 1 to 15) until the day before the necropsy through the mating period (42 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 4 of lactation (day of delivery was Day 0 of lactation) through the mating and pregnancy periods and delivery. One female which did not deliver was kept until day before the necropsy.
Frequency of treatment:
Parental males and females daily by oral gavage.
Remarks:
0, 110, 330 and 1000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- A total of 106 rats were used on study (48 males and 58 females).
- Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg groups in the recovery test) and 12 females (5 females
each were added for the control and 1000 mg/kg groups in the recovery test).
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: A preceeding 14 day repeated dose oral study with the test substance to rats, indicated that at a high dose of 1000 mg/kg/day would be expected to provide any obvious toxicological changes.

- Post-exposure recovery period in satellite groups: Yes in males and females.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were performed once a day before commencement of dosing, then once a week during the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36, and 42. The recovery males were also weighed on Days 43, 50, and 56. The satellite females were weighed on the same manner as the recovery males. The test females were weighed on
Days 1, 8, and 15, Days 0, 7, 14, and 20 of gestation, and Days 0 and 4 of lactation. The final body weight was also measured on the day of the scheduled necropsy.

FOOD CONSUMPTION: YES
- Food consumption was measured for Days 1 to 8, 8 to 15, 29 to 36, and 36 to 40 in test and recovery males, and for Days 43 to 50 and 50 to 54 in recovery males only. For females, it was measured fro Days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 in satellite females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 for test males, and Day 57 for the recovery males and satellite females, and Day 5 of lactation for the test females.
- Anaesthetic used for blood collection: Yes; intraperitoneal injection of sodium pentabarbitol (30 mg/kg).
- Animals fasted: Yes; for 19-23 hours before sampling.
- How many animals: 5 animals for the test males, all recovery males and satellite females, and 5 females from the earlier partuition date.
- Parameters examined: Leukocyte, RBC, HGB, Haematocrit, MCV, MCH, MCHC, reticulocyte ratio and count, differential leukocyte ratio/count, PT and APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Straight after sampling for haematology.
- Animals fasted: Yes; for 19-23 hours before sampling
- How many animals: 5 animals for the test males, all recovery males and satellite females, and 5 females from the earlier partuition date
- Parameters examined: Total protein, albumin, A/G ratio), ASAT, ALAT, GGT, ALP, total bilirubin, total bile acids, total cholesterol, triglycerides, glucose, urea nitrogen, creatinine, calcium, inorganic phosphorus, sodium, potassium and chloride.

URINALYSIS: Yes (In males)
- Time schedule for collection of urine: 5 males in the final dosing week (week 40).
- Animals fasted: Yes (water was available).
- Parameters examined: pH, protein, glucose, ketone and occult blood all analysed using a reagent strip. As no abnormalities related to treatment were detected in any of the parameters analysed, the urinary sediment and accumulated urine test and the urinalysis during the recovery period was not conducted.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The functional test and motor activity measurements were observed in 5 males per group once in Week 6. For females, 5 dams from the nearer parturition date were selected from each group and observed once during the lactation period. The functional test and motor activity observations were not measured during the recovery periods, as no treatment related changes were noted during the dosing periods.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity to stimuli / grip strength / motor activity.
Sacrifice and pathology:
All animals were sacrificed by exsanguination under anaeshesia and subjected to necropsy (after sampling for haematology and blood chemistry investigations).
Samples of the following tissues/organs were preserved in 10% phosphate buffered formalin (testes and epididymides in Bouin's fixative and the eyes in a davidson's solution):
- Brain (including cerebrum, cerebellum, medulla oblongata and pons).*
- Pituitary
- Spinal cord (cervical)
- Eyes
- Submandibular lymph node
- Submandibular gland
- Thyroid*
- Parathryoid
- Thymus*
- Trachea
- Lungs (including bronchus)
- Heart*
- Stomach
- Duodenum
- Jujunum
- Ileum (including payer's patch)
- Cecum
- Colon
- Rectum
- Mesenteric lymph node
- Liver*
- Pancreas
- Spleen*
- Kidney*
- Adrenal*
- Urinary bladder
- Testis*
- Epididmymis*
- Seminal vesicle (including dorsolateral and coagulating gland)*
- Prostate (ventral)*
- Ovary*
- Uterus
- vagina
- Femur
- bone marrow (femur)
- Sternum
- Bone marrow (sternum)
- M.Biceps femoris
- sciatic nerve
- Other gross legions
Tissues marked (*) were also weighed

Histopathology
Tissues from 5 high dose and control animals were processed and stained with haematoxylin and eosin prior to microscopic examination. One non-pregnant female also had the ovaries stained an examined. As no test substance related changes were observed at 1000 mg/kg, the additional microscopic examination of the intermediate groups was not performed.
Statistics:
Histopathological findings were analysed using Fisher's exact probability test. Copulation index, fertility index, gestation index, delivery index and sex ratio were analysed using the Chi-squared test. Implantation index, stillborn rate, external anomaly index, external anomaly typing index, live birth index, and viability index were analysed using Wilcoxon's rank sum test.

Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis (reagent strip), hematology, blood chemistry, absolute and relative organ weight, estrous cycle, number of estrus, days until copulation, gestation length, number of corpora lutea, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes) were initially analysed using Bartlett's test. When the data was homogenous the Dunnet's multiple comparison test was applied, and when not homogenous, Steel's multiple comparison test was applied for the control group and each test group. For the urinalysis reagent strip, Steel's test was performed.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
In males only
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No deaths occured during study and no abnormal clinical signs were observed throughout the dosing and recovery periods.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant differences were observed in any sex in the treatment-groups compared with the controls.

FOOD CONSUMPTION: No statistically significant differences were observed in any sex in the treatment-groups compared with the controls.

HAEMATOLOGY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence and changes were not seen at the end of the dosing period.

CLINICAL CHEMISTRY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence, changes were not seen at the end of the dosing period, or values were similar to historical control values.

URINALYSIS: In the qualitative analysis, no statistically significant difference was observed in males in the treated-groups as compared to the control group.

NEUROBEHAVIOUR: There were considered not to be any treatment related changes with regards to reactivity to stimuli, grip strength and motor activity in males or females at any treatment group. No treatment related changes were also observed in the detailed clinical investigations (hand-held or open field observation). Changes observed during the motor activity investigations, were not considered treatment related as no dose-repsonse was observed.

ORGAN WEIGHTS: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods. Any effects observed were deemed to be non-dose dependant in their incidence, changes were not seen at the end of the dosing period and/or there was no associated histopathological findings.

GROSS PATHOLOGY: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods, including the non-pregnant female. Any effects observed were deemed to be non-dose dependant in their incidence.

HISTOPATHOLOGY: NON-NEOPLASTIC: No treatment related changes were observed in any treatment group at the end of the administration or recovery periods, including the non-pregnant female. Any effects observed were deemed to be non-dose dependant in their incidence.

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested
Key result
Critical effects observed:
no
Conclusions:
The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day in both sexes under the conditions of this study, as there were no effects of parameters associated with repeated dose toxicity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

A reliable combined repeated dose and reproductive screening study is available detailing the NOAEL for repeated dose toxicity (oral exposure) as 1000 mg/kg/day in both sexes under the conditions of this study, as there were no effects of parameters associated with repeated dose toxicity. No specific target organ effects were detailed based on findings on study including a lack of treatment related pathological changes.

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