Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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:
2016-08-25 to 2017-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-25 to 2017-02-22
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)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects of toxicological significance were observed.
Key result
Critical effects observed:
no
Conclusions:
This study was conducted to evaluate the potential toxicities of the test substance in the general systemic and reproductive/development when administered via oral gavage to Sprague-Dawley rats with dose levels of 0, 100, 300 and 1000 mg/kg. There were no test item-related adverse effects in general effects and reproduction/development up to 1000 mg/kg. Therefore, the No Observed Adverse Effect Levels (NOAELs) for general toxicity effects and reproduction/developmental toxicity are considered to be at least 1000 mg/kg/day.
Executive summary:

This study was conducted to evaluate the potential toxicities of a test substance, 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, 100, 300 and 1000 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 (at least 50 days), and continued through the lactation day 13 in females. Additional animals in the recovery groups 0 and 1000 mg/kg (6 animals per sex per group) received the test substance, but were not mated. Afterwards, they were assigned to 2 weeks of recovery period after the completion of the test item administration.

No deaths or moribund animals occurred in any group throughout the study. Test item-related salivation was observed in both sexes at 1000 mg/kg; however, it was not considered to have toxicological relevance since it was considered to be attributed to the palatability of the test item. At 1000 mg/kg, test item-related changes were also observed including increased prothrombin time (up to 1.11-fold of the control) in both sexes and increased kidney weight (1.13-fold of control) in females. However, it was not considered to have toxicological relevance since there was no correlated microscopic findings. In addition, in males in all test item-treated groups, decreased gamma glutamyl transpeptidase (up to 32% of control) was observed. However, it was not considered to have toxicological relevance since there was no correlated microscopic finding.

No test item-related change was observed in oestrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examinations. Test item-related increase in T4 was observed in adult males (1.14-fold of control) and pups (1.20-fold of control) at 1000 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 item-related adverse effects for general toxicity effects and reproductive/developmental effects were observed up to 1000 mg/kg. Therefore, the No Observed Adverse Effect Levels (NOAELs) for general toxicity effects and reproduction/developmental toxicity are considered to be at least 1000 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)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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)
- Weight at study initiation: (P) Males: 315.5-410.9 g, Females: 220.5-270.5 g
- Fasting period before study: not specified
- Housing: stainless-steel cage (255W×465L×200H mm) - 2 animals per cage (acclimation, pre-treatment, treatment, mating (1 (male) and 1 (female)) 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: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1-24.1°C
- Humidity (%): 39.8-65.4%
- Air changes (per hr): 10-20 times/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Administration / exposure

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. The required amount of the test item was weighed and suspended in corn oil with a stirrer for approximately 5 min to prepare the target concentration. Formulation for the high dose group was prepared first and then lower dose formulations were prepared by diluting the higher dose formulation with corn oil

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
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or 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 for homogeneity analysis were also analysed for verification of dose level concentration. Results of dose formulations were 101.34, 100.97 and 105.60% at each dose levels of 100, 300 and 1000 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 (at least 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:
The control item or dose formulations were administered by oral gavage once a day at approximately the same time each day (08:30 - 13:00) for 7 days/week.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
18 (vehicle control), 12 (100 and 300 mg/kg bw/day), 18 (1000 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 repeated dose 2-week dose-range-finding study in Sprague-Dawley rats in which doses of 60, 250 and 1000 mg/kg have been tested. In that study, no treatment-related changes in all examined items were observed in any doses tested. Based on these results, 1000 mg/kg which is a limit dose level was selected as the high dose, and 300 and 100 mg/kg are selected as the middle and low doses. Control animals were administered with corn oil.
- Rationale for animal assignment (if not random): Animals were selected for use 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 and pre-treatment periods. They were randomised and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

Examinations

Parental animals: Observations and examinations:
MORTALITY: Mortality and morbidity observations were conducted twice daily except for the acclimation period. In addition, observations were conducted 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 (parturition completed females were observed additionally once more)); 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 6 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: 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)

MATING:
- Time schedule for examinations: Mating confirmation was checked every morning during the mating period and was confirmed with vaginal plug(s) and/or sperm in the vaginal smear

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.
- Dams were allowed to litter. The duration of gestation, the number of dead and live pups, runts, sexing of live pups, individual body weight of live pups and external abnormalities were observed.
- Females which were not observed to have parturition were sacrificed after GD 27.

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 haemoglobin, mean corpuscular haemoglobin concentration, platelet count, reticulocyte counta, white blood cell 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, alanine aminotransferase, total bilirubin, alkaline phosphatase, gamma glutamyl transpeptidase, creatine phosphokinase, calcium, inorganic phosphorus, sodium, potassium, chloride.

THYROID HORMONE ANALYSIS
- Blood collection: lactation day 13 (all dams) and 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 were taken at termination to examine the stage of the oestrus 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 internal 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 euthanised on the day after final dosing and necropsied.
- Maternal animals: All surviving main group animals on lactation day 14 were euthanised.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
All tissues collected from main group animals were further processed to slides, stained with hematoxylin and eosin, and examined microscopically.

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. The tissues were placed in the appropriate fixative for approximate 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.

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 euthanised 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 from non-pregnant females until mating were included in the report. Data were 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 were analysed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were assessed using Dunnett’s Test. Heterogeneous data were analysed 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 were analysed for homogeneity for variance using F-test. Homogeneous data were analysed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test. Heterogeneous data were analysed using Kruskal-Wallis Test and significant differences between control and recovery group were assessed using Dunn’s Rank Sum Test.

One-way analysis of covariance (ANCOVA) was used to analyse pup body weight. The litter size was used as the covariate. Litter data were statistically evaluated using the statistical unit as a litter. Data which were presented as frequencies were analysed 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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed in 10 and 9 animals in males and females at 1000 mg/kg, respectively. 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 either sex, of all groups throughout the study.

Unscheduled sacrifice was conducted on one female in vehicle control group on lactation day 3 because all pups were found dead. It was considered incidental since it was observed in vehicle control group and there was no change in clinical signs and macroscopic findings in this unscheduled sacrificed animal.

Non-parturition was observed in 1 female in each dose group (100, 300 and 1000 mg/kg). Unscheduled sacrifice was conducted on gestation day 28 in such females. It was considered incidental since there was no test item-related changes in macroscopic and microscopic findings.
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.

A statistically significant change in body weight gain during the study was not considered test item-related since it was transient.
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.

A statistically significant change in food consumption during the study was not considered test item-related since it was not dose-dependent and/or transient.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes in haematology were observed in both sexes during the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in gamma glutamyl transpeptidase (GGT, up to 32% of control) in males in all test item-treated groups was observed. It was considered test item-related but not considered adverse as there was no correlated microscopic findings. This change was recovered after the recovery period.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in functional behaviour were observed in both sexes during the study.

A statistically significant decreased grip strength of hind limb in males at all test item-treated groups was not considered test item-related since there was no change in grip strength of forelimbs, no findings in recovery males, and no findings in regard to the same parameters in females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in absolute weight of kidneys (1.13-fold of control) was observed in females at 1000 mg/kg. It was considered test item-related but not considered adverse as there was no correlated microscopic findings. This change was also observed after recovery period; however, it was not considered adverse since this change remained within the historical control data.

Other statistically significant changes in organ weights were not considered test item-related since they were not dose-dependent and there was no correlated 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 as they are commonly seen in this animal strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in microscopic findings were observed in both sexes during the study.

Microscopic findings observed during this study were considered to be incidental or spontaneous since they were infrequent, generally of low severity and similarly distributed among control and test item-treated groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in both sexes during the study.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity examination:
- No test item-related changes in motor activity examination were observed in both sexes during the study. A statistically significant change in motor activity parameters during the study was not considered test item-related since it was transient or only observed in females. The change in females considered incidental since the control values were much lower when compared with the historical control data.

Coagulation:
- A statistically significant increase in prothrombin time (PT, up to 1.11-fold of control) was observed in both sexes at 1000 mg/kg. It was considered test item-related but not considered adverse as there were no correlated microscopic findings. This change was recovered after the recovery period.

Thyroid Hormone (T4) Analysis:
- A statistically significant increase in thyroid hormone (T4) was observed in adult males (1.14-fold of control) at 1000 mg/kg. These changes were considered test-item related but non-adverse since there were no correlated changes in other parameters including microscopic findings in thyroids (with parathyroids).

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in oestrus cycle were observed during the study.

A statistically significant decreased length of oestrus cycle in all test item-treated groups was not considered test item-related since it was within the normal biological range.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test item-related changes in precoital time, fertility data and reproductive and littering findings were observed during the study.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects of toxicological significance were observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related changes in F1 pups were observed during the study.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Perinatal death: 1/2/1/0 (VC/ 100/ 300/ 1000)
Dead Pups on PND 0-4 (N): 9/10/0/4 (VC/ 100/ 300/ 1000)
Dead Pups on PND 5-13 (N): 1/0/0/0 (VC/ 100/ 300/ 1000)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in F1 pups were observed 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 pups were observed during the study.

No test item-related changes in F1 culled pups external examination were observed during the study. One pup at 100 mg/kg exhibited a loss of tail but this was not considered test item-related since it was not dose-dependent and the incidence was low.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone (T4) analysis:
- A statistically significant increase in thyroid hormone (T4) was observed in pups (1.20-fold of control) at 1000 mg/kg. These changes were considered test-item related but non-adverse since there were no correlated changes in other parameters including microscopic findings in thyroids (with parathyroids).

F1 pups anogenital distance:
- No test item-related changes in F1 pups were observed during the study.

F1 male pups nipple retention:
- No test item-related changes in F1 male pups nipple retention were observed during the study. A slight increase in male pups nipple retention was observed in all test item-treated groups, but was not considered test item-related since it was within the historical control data.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 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.

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)

Applicant's summary and conclusion

Conclusions:
This study was conducted to evaluate the potential toxicities of the test substance in the general system and reproductive/development when administered via oral gavage to Sprague-Dawley rats with dose levels of 0, 100, 300 and 1000 mg/kg. There were no test item-related adverse effects in general effects and reproduction/development up to 1000 mg/kg. Therefore, the No Observed Adverse Effect Levels (NOAELs) for general toxicity effects and reproduction/developmental toxicity are considered to be at least 1000 mg/kg/day.
Executive summary:

This study was conducted to evaluate the potential toxicities of a test substance, 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, 100, 300 and 1000 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 (at least 50 days), and continued through the lactation day 13 in females. Additional animals in the recovery groups 0 and 1000 mg/kg (6 animals per sex per group) received the test substance, but were not mated. Afterwards, they were assigned to 2 weeks of recovery period after the completion of the test item administration.

No deaths or moribund animals occurred in any group throughout the study. Test item-related salivation was observed in both sexes at 1000 mg/kg; however, it was not considered to have toxicological relevance since it was considered to be attributed to the palatability of the test item. At 1000 mg/kg, test item-related changes were also observed including increased prothrombin time (up to 1.11-fold of the control) in both sexes and increased kidney weight (1.13-fold of control) in females. However, it was not considered to have toxicological relevance since there was no correlated microscopic findings. In addition, in males in all test item-treated groups,decreased gamma glutamyl transpeptidase (up to 32% of control) was observed. However, it was not considered to have toxicological relevance since there was no correlated microscopic finding.

No test item-related change was observed in oestrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examinations. Test item-related increase in T4 was observed in adult males (1.14-fold of control) and pups (1.20-fold of control) at 1000 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 item-related adverse effects for general toxicity effects and reproductive/developmental effects were observed up to 1000 mg/kg. Therefore, the No Observed Adverse Effect Levels (NOAELs) for general toxicity effects and reproduction/developmental toxicity are considered to be at least 1000 mg/kg/day.