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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-04 to 2017-02-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity and Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-04 to 2017-02-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Specific Pathogen Free
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc., 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do, 13201, Republic of Korea
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks (males), 12 wks (females); (F1) x wks
- Weight at study initiation: (P) Males: 329.5-390.7 g; Females: 213.3-264.2 g; (F1)
- Fasting period before study: not specified
- Housing: stainless-steel cage (255W×465L×200H mm) - 2 animals per cage (acclimation, pre-treatment, treatment, mating and post-mating)
poly sulfone cages (260W×420L×180 mm) - 1 mated female during gestation, 1 dam with pups during lactation
stainless-steel cage (255W×465L×200H mm) - 2 animals per cage (recovery)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 24.1°C
- Humidity (%): 42.3 - 64.4%
- Air changes (per hr): 10 - 20 times/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose was based on the most recently measured body weight. Animals were dosed at a volume of 2 mL/kg. Dose formulation was continuously stirred by magnetic stirrer in the animal room (before and throughout the dosing procedure).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Lot/batch no. (if required): MKBW9504V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: 2 per cage (post-mating); then 1 mated female per cage (gestation) and 1 dam with pups per cage (lactation)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted based on the method used in a previous study conducted by the testing laboratory and samples of dose formulations were 98.56, 97.59 and 106.50% at each dose levels of 75, 250 and 800 mg/kg. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
Dosing of the males began 14 days prior to mating and continued throughout mating prior to sacrifice (total 50 days). Dosing of the females began 14 days prior to mating and continued throughout lactation day (LD) 13. Animals of the recovery group were not mated and assigned to 2 weeks of recovery period after the completion of administration.
Frequency of treatment:
Once a day at approximately the same time each day (08:30 - 13:00) for 7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
18 (vehicle control), 12 (75 and 250 mg/kg bw/day), 18 (800 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose level was selected based on the results of a 2-week dose range-finding study in Sprague-Dawley rats in which doses of 60, 250 and 1000 mg/kg have been tested. In males at 1000 mg/kg, a test item-related decreased body weight gain (71% of control) was observed. In addition, test item related salivation was observed in both sexes at 250 and 1000 mg/kg. Based on the result of this dose range-finding study, 800 mg/kg was selected as the high dose that is anticipated to produce minimal toxicity. 250 and 75 mg/kg were selected as the middle and low dose. Control group was administered with vehicle.
- Rationale for animal assignment (if not random): Animals were selected for use in study on the basis of adequate body weight (recorded on the day of receipt and group assignment), estrus cycle and free from clinical signs of disease or injuries during the acclimation (5 days) and pre-treatment (14 days) periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.
Parental animals: Observations and examinations:
MORTALITY: Mortality and morbidity observations were conducted twice daily except for the acclimation period and once on necropsy day.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day (acclimation and pre-treatment), twice a day (before and after dosing; treatment, mating, post-mating, gestation, lactation); once a day (recovery), once prior to necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week in all animals

BODY WEIGHT: Yes
- Time schedule for examinations: animal receipt day and day 5 of acclimation; once per week (pre-treatment); first day of dosing and then once per week (treatment); once per week (mating, post-mating); gestation days 0, 7, 14 and 20; lactation days 0, 4, 13; once per week (recovery)

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
- Time schedule: once per week (treatment and post-mating); gestation day 0, 7, 14 and 20; lactation day 0, 4 and 13; once per week (recovery)

FUNCTIONAL OBSERVATIONS:
- Sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity were conducted in 6 males and 6 females, selected from each group and in all animals of the recovery group shortly before scheduled sacrifice.

PARTURITION MONITORING:
-Mating-proven females were monitored twice daily for signs of parturition including abortion, premature delivery and difficult or prolonged parturition from GD 21.

CLINICAL PATHOLOGY
- Blood collection: 6 males and 6 females from each group prior to terminal sacrifice and animals of the recovery group.
- Haematology parameters examined: haematology (total leukocyte count, total red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, prothrombin time, fibrinogen, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte counta, WBC differential count, Activated partial thromboplastin time)
- Clinical chemistry parameters examined: glucose, blood urea nitrogen, creatinine, total protein, albumin, albumin/globulin ratio, total cholesterol, triglyceride, phospholipid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), creatine phosphokinase, calcium, inorganic phosphorus, sodium, potassium, chloride

THYROID HORMONE ANALYSIS
- Blood collection: lactation day 13 (all dams), at termination (all adult males)
Estrous cyclicity (parental animals):
A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle during the treatment period until mating was examined.

In addition, vaginal smear of sacrificed females was taken at termination to examine the stage of the estrus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
mortality (once per day), general clinical signs including general appearance and behaviour changes (once per day), body weight and sex (post-natal day 0, 4 (before culling) and 13), anogenital distance (post-natal day 4), number of nipples in all male pups (post-natal day 12), thyroid hormone analysis (blood collection on post-natal day 4 - at least two culled pups, one male and one female if possible, per litter - and lactation day 13 - at least two pups, one male and one female if possible, per litter)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and visceral abnormalities; after day 5 of lactation, dead pups were necropsied with special attention to all vital organs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanized on the day after final dosing and necropsied.
- Maternal animals: All surviving main group animals on lactation day 14 were euthanized.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the abdominal, thoracic and cranial cavities. Organs were removed and examined; special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed for all animals at terminal and recovery sacrifice and organ/body weight ratios using the terminal body weight (TBW) obtained prior to necropsy was calculated. Paired organs were weighed together unless gross abnormalities are present. However, paired reproductive organs were weighed separately:
Testes (right, left, a), brain (b), prostate (b), kidneys (b), spleen (b), heart (b), thymus (b), ovaries (right, left, b), epididymides (right, left, a), pituitary gland (b), liver (b), adrenal glands (b), lung (b), uterus (with cervis, b), seminal vesicles (with coagulation gland (b).
a: All male animals
b: Six animals were selected in each group and sex.

The following tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative. All tissues collected from main group animals were further processed to slides, stained with hematoxylin and eosin, and examined microscopically:
Ovaries (a), prostate (a), testes (a), epididymides (a), uterus (with cervix, a), seminal vesicles (with coagulation gland, a), alterations (a), thyroids (with parathyroids, a,c), brain (b), eye (b), spinal cord (cervical, thoracic, lumbar, b), trachea (b), stomach (b), lung (with bronchi, b), cecum (b), urinary bladder (b), skeletal muscle (b), vagina (b), colon (b), mandibular lymphnode (b), duodenum (b), mesenteric lymphnode (b), rectum (b), sciatic nerve (b), ileum (b), femur with marrow (F-T joint, b), jejunum (b), spleen (b), liver (b), heart (b), kidneys (b), thymus (b), adrenal glands (b).
a: All animals
b: Six animals were selected in each group and sex.
c: Thyroids (with parathyroids) for adult males and pups were processed for microscopic findings to clarify the change of thyroid hormone (T4). Parathyroids were examined only if present in the routine section.
d: Collected but not prepared for histopathology
Postmortem examinations (offspring):
SACRIFICE
- All pups were euthanized on post-natal day 13.

GROSS NECROPSY
- Gross necropsy consisted of external examinations for abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroids (with parathyroids) were preserved in 10% neutral buffered formalin for each one male and one female pup (blood sampling pups) per litter, if possible.
Statistics:
Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System or Statistical Analysis Systems. Data was considered to be significant when p<0.05 or p<0.01.

Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data was analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.

For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test. Homogeneity data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.

One-way analysis of covariance (ANCOVA) was used to analyze pup body weight. The litter size was used as the covariate. Litter data was statistically evaluated using the statistical unit as a litter.

Data presented as frequencies was analyzed by ¿2-test followed by the Fisher's exact test where necessary.
Reproductive indices:
Based on the results, the following reproduction indices were calculated:
Mating Index (%)
= (No. of males with evidence of mating/No. of males paired) × 100
= (No. of females with evidence of mating/No. of females paired) × 100

Fertility Index (%)
= (No. of males impregnating a female/No. of males paired) × 100
= (No. of pregnant females/No. of females paired) × 100

Fecundity Index and Pregnancy Index (%)
= (No. of males impregnating a female/No. of males with evidence of mating) × 100
= (No. of pregnant females/No. of females with evidence of mating) × 100

Precoital Time: No. of days taken to mate
Offspring viability indices:
Based on the results, the followings were calculated.
On Day 0 of Lactation

Delivery Index = No. of dams with live pups / No. of pregnant dams × 100
On Day 4 of Lactation

Viability Index = No. of live pups on Day 4 of lactation / No. of live pups at birth × 100
On Day 13 of Lactation

Weaning Index = No. of live pups on Day 13 of lactation / No. of pups on Day 4 of lactation after culling × 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed in both sexes at 800 mg/kg. It was considered test item-related but not toxicologically relevant since it was considered to be attributed to the palatability of the test item.

Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have a dose-response.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths or moribund animals occurred in both sexes of all groups throughout the study.

Unscheduled sacrifice was conducted on one female at 800 mg/kg on GD 24 because all pups were found dead. This animal was also observed to have prolonged parturition, irregular respiration and skin paleness during GD 23 to 24. Macroscopic observations showed greenish black luminal contents in stomach and colon and pinkish transparent thoracic fluid. This finding was was not considered to be of toxicological relevance since no test item-related adverse effects in other parameters at 800 mg/kg were observed during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight and body weight gain were observed in both sexes during the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes in food consumption were observed in both sexes during the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in haematology were observed in both sexes during the study.

A statistically significant change in haematology was not considered test item-related since these changes were minimal, there was no dose-dependency and there was no correlation with microscopic findings.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related changes in clinical chemistry were observed in both sexes during the study.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related changes in functional behavior examination were observed in both sexes during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in organ weights were observed in both sexes during the study.

A statistically significant change in organ weights was not considered test item-related since these changes were minimal and there was no correlation with microscopic findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in macroscopic findings were observed in both sexes during the study.

Macroscopic findings observed during this study were considered to be incidental or spontaneous that commonly seen in this animal strain given their low incidence or severity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related changes in microscopic findings were observed in both sexes including F1 pups thyroids (with parathyroids) during the study.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity examination:
- A statistically significant change in distance, speed and freezing time in males at 75 mg/kg was not considered test item-related due to the lack of dose-dependency.

Coagulation:
- No test item-related changes in coagulation were observed in both sexes during the study.

Thyroid Hormone (T4) Analysis:
- A statistically significant increase in thyroid hormone (T4) was observed in adult males (1.24-fold of control) at 800 mg/kg (p < 0.01). These changes were considered test-item related but non-adverse since there were no correlated changes in other parameters including microscopic findings of thyroids (with parathyroids).
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
No test item-related changes in estrus cycle were observed during the study.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects were observed regarding precoital time, fertility data, and/or reproductive and litter findings during the study.
No test item-related changes in reproductive and littering findings were observed during the study.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects of toxicological significance were observed
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related changes in F1 pups clinical signs were observed in both sexes during the study.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Perinatal death: 0/0/1/1 (VC/ 75/ 250/ 800)
Dead Pups on PND 0-4 (N): 2/2/4/2 (VC/ 75/ 250/ 800)
Dead Pups on PND 5-13 (N): 0/1/0/0 (VC/ 75/ 250/ 800)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in F1 pups body weight were observed in both sexes during the study.
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related changes in F1 culled pups external examination were observed in both sexes during the study.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups anogenital distance:
- No test item-related changes in F1 pups anogenital distance were observed in both sexes during the study. A statistically significant decrease in anogenital distance in females at 75 mg/kg was not considered test-item related since it was not dose-related.

F1 male pups nipple retention:
- No test item-related changes in F1 male pups nipple retention were observed during the study.

Thyroid hormone (T4) analysis:
- A statistically significant increase in thyroid hormone (T4) was observed in pups (up to 1.22-fold of control) at 250 and 800 mg/kg (p < 0.05 and p < 0.01, respectively). These changes were considered test-item related but non-adverse since there were no correlated changes in other parameters including microscopic findings of thyroids (with parathyroids).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects of toxicological significance were observed
Key result
Reproductive effects observed:
no
Conclusions:
This study was conducted to evaluate the potential toxicity of 3 -methylbutyl isovalerate on systemic toxicity and reproductive/development toxicity when administered via oral gavage to Sprague-Dawley rats at dose levels of 0, 75, 250 and 800 mg/kg bw/day. There were no test item-related adverse systemic toxicity effects or reproduction/development effects up to 800 mg/kg bw/day. Therefore, the No-Observed-Adverse-Effect Levels (NOAELs) for general toxicity effects and reproduction/developmental toxicity are considered to be at least 800 mg/kg bw/day.
Executive summary:

This study was conducted to evaluate the potential toxicities of the test substance 3 -methylbutyl isovalerate regarding general systemic effects and reproductive/developmental toxocity. The test substance was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 75, 250 and 800 mg/kg with a dose volume of 2 mL/kg. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (total 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in the recovery groups 0 and 800 mg/kg (6 animals per sex per group) received the test item but were not mated. Afterwards, they were assigned to 2 weeks of recovery period after the completion of test item administration. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and evaluated. In addition, reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were measured. Thyroid hormone (T4) level in blood was also analysed for adult males and pups at sacrifice.

No deaths or moribund animals occurred in any group throughout the study. One female of which all pups were found dead and which showed prolonged parturition, irregular respiration and skin paleness at 800 mg/kg was sacrificed unscheduled on gestation day (GD) 24.The relationship of test item administration and these findings was uncertain; however, they were not considered to have toxicological relevance since no test substance -related adverse effects in other parameters at 800 mg/kg were observed during the study.

No test substance-related change was observed up to 250 mg/kg. At 800 mg/kg, test item-related salivation was observed in both sexes but was not considered to have toxicological relevance. No test substance -related change was observed in body weight, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings.

In reproductive/developmental observations, no source substance-related changes were observed in estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination.

Test substance-related increase in T4 was observed in adult males (1.24-fold of control) at 800 mg/kg and in pups (up to 1.22-fold of control) at 250 and 800 mg/kg. However, it was not considered to have toxicological relevance since there were no correlated changes in other parameters including microscopic findings of thyroids (with parathyroids).

In conclusion, no test substance-related adverse effects in general effects and reproduction/development were observed up to 800 mg/kg. Therefore, the No-Observed-Adverse-Effect Levels (NOAELs) for general toxicity and reproduction/developmental toxicity are considered to be at least 800 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects of toxicological significance were observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
This study was conducted to evaluate the potential toxicity of the test substance on systemic toxicity when administered via oral gavage to Sprague-Dawley rats at dose levels of 0, 75, 250 and 800 mg/kg bw/day. There were no test item-related adverse systemic toxicity effects up to 800 mg/kg bw/day. Therefore, the No-Observed-Adverse-Effect Levels (NOAELs) for general toxicity is considered to be at least 800 mg/kg bw/day
Executive summary:

This study was conducted to evaluate the potential toxicities of the test item regarding general systemic effects and reproductive/developmental toxicity. The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 75, 250 and 800 mg/kg with a dose volume of 2 mL/kg. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (total 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in the recovery groups 0 and 800 mg/kg (6 animals per sex per group) received the test item but were not mated. Afterwards, they were assigned to 2 weeks of recovery period after the completion of test item administration. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and evaluated. In addition, reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were measured. Thyroid hormone (T4) level in blood was also analysed for adult males and pups at sacrifice.

 

No deaths or moribund animals occurred in any group throughout the study. One female of which all pups were found dead and which showed prolonged parturition, irregular respiration and skin paleness at 800 mg/kg was sacrificed unscheduled on gestation day (GD) 24.The relationship of test item administration and these findings was uncertain; however, they were not considered to have toxicological relevance since no test item-related adverse effects in other parameters at 800 mg/kg were observed during the study.

 

No test item-related change was observed up to 250 mg/kg. At 800 mg/kg, test item-related salivation was observed in both sexes but was not considered to have toxicological relevance. No test item-related change was observed in body weight, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings.