Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2017 - 11 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control database. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ORIENTBIO INC., Republic of Korea
- Age at study initiation: male: 9 weeks; female: 9 weeks
- Weight at study initiation: male: 301–331 g; female: 198-237 g
- Fasting period before study: no
- Housing: Animals per cage: 1-2 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm) and Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum, pelleted rodent chow (Teklad Certified Irradiated Global 18 % Protein Rodent Diet 2918C)
- Water: ad libitum, public tap water, filtered and irradiated by ultraviolet light
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 26.7
- Humidity (%): 40.8 - 69.6
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed using electronic balances, placed in a container, mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (4.8–7.1 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: based on preliminary solubility test
- Concentration in vehicle: 0.5 and 500 mg/mL
- Amount of vehicle: 2 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: single
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations was conducted using a Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle layer of each dosing formulation prior to dosing and analyzed for verification of dose level concentration.
Duration of treatment / exposure:
Males of the main group were dosed for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 22 days of post-mating).
Males and females of the recovery groups were dosed for 49 days.
Females of the main group were dosed for 2 weeks prior to mating until Postpartum Day 13. Females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals were not mated.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 12
Recovery groups: 6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 2-week repeated oral dose range finding study
Positive control:
No positive control was conducted.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: general condition, clinical signs, moribundity, mortality and females also signs of abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing, once weekly for the dosing and recovery periods
- Detailed clinical observations included: skin, fur, eyes, mucous membranes, occurrence of secretion and excretion, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern etc.), changes in gait, posture and response to handling, and the presence of clonic or tonic movements, stereotypy (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: males of main group and males/females of recovery group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was recorded, individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 h
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, 18 h
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but free access to drinking water
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: 6 males and 6 females were randomly selected from the main groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength test, motor activity

IIMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter (if available above culling target), all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (Total T4) [ug/dL] and Thyroid stimulating hormone (TSH) [μUI/mL], Method: Chemiluminescent competitive immunoassay,
Oestrous cyclicity (parental animals):
The estrous cycle for females of the main groups was observed from dosing initiation day to confirmed copulation day and on the necropsy day. Smears of vaginal mucosa were prepared in the morning daily. Prepared smears of the vaginal mucosa were stained and examined using light microscopy. The estrous cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrous cycle was defined as the period between the day of estrus and the day prior to next estrus. Estrous cycle was calculated from dosing initiation day to the day before mating initiation.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals of the main groups were sacrificed on Day 50. All animals of the recovery groups were sacrificed two weeks after final dosing.
- Maternal animals: All surviving animals of the main groups were sacrificed on Postpartum Day 14. Non-copulated females were sacrificed on Day 26 after the last day of mating. Non-pregnant females were sacrificed on Gestation Day 26. All animals of the recovery groups were sacrificed two weeks after final dosing.

Animals that died during the study period:
Necropsies were conducted on all animals found dead as soon as possible. When not feasible, they were stored under refrigeration until necropsy. Gross pathology findings were recorded and tissue samples were collected for histopathology.

GROSS PATHOLOGY: Yes. All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external surfaces and internal organs.

HISTOPATHOLOGY: Yes, organs listed in table 4 were prepared for histopathology for all animals of the main groups and all animals of the recovery groups. The testis and eyes with optic nerve will be fixed in Davidson’s fixative. All other tissues will be preserved in 10% neutral buffered formalin. The thyroids from all male and female pups per litter, sacrificed on PND 13 or shortly thereafter, will be preserved in 10% neutral buffered formalin. Organs listed in Table No. 5 were prepared for histopathology.

ORGAN WEIGHTS:
The organs listed in table 5 were weighed from all males and females in the main groups and recovery groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at PND 13.
- These animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS: no
Statistics:
The statistical analysis of this study were conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).
For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, litter size, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters and organ weights, grip strength, motor activity, Bartlett’s test will be conducted to analyze for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test will be applied on homogeneous data, then, if significant (significance level: 0.05), Dunnett’s t-test will be applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test will be applied on heterogeneous data, then, if significant (significance level: 0.05), Steel’s test will be applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of sensory function, mating index, fertility index and other data associated with gestation will be analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery groups, Folded-F test will be employed to analyze for homogeneity of variance (significance level: 0.05, two-tailed). Student t-test will be applied for homogeneous data, but if overruled, Aspin-Welch t-test will be applied (significance levels: 0.05 and 0.01, two-tailed).
Reproductive indices:
Mating index, mating period, gestation period, male and female fertility index and gestation index were calculated.
Offspring viability indices:
Mean litter size, live birth index, viability index on postnatal day 0 and 4 and sex ratio were calculated.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dead animals:
In the main group, one female in the 0 mg/kg/day group and 3 females in the 800 mg/kg/day group died. One dam in the 0 mg/kg/day group was found dead on PPD 1 after showing dystocia and delayed delivery. The dystocia observed at 0 mg/kg/day was considered to be incidental. Three pregnant females in the 800 mg/kg/day group were found dead before or during parturition. Salivation was observed in these three animals at 800 mg/kg/day. In the recovery group, one female in the 0 mg/kg/day group was found dead on Day 60 without any abnormal clinical signs. Since there were no deaths at 800 mg/kg/day in the recovery group, the death was considered as test substance-related reproductive toxicity.

Surviving animals:
All males of the main and recovery groups survived the duration of the study. In the main group, salivation was observed in four males and seven females at 800 mg/kg/day. In the recovery group, salivation was observed in five males and six females at 800 mg/kg/day. However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Mass in the chest was observed in one female at 50 mg/kg/day. The
mass was microscopically identified as mammary gland adenocarcinoma. It was considered to be occurred spontaneously since it showed low incidence and no dose dependency. At 800 mg/kg/day, soft stool was temporarily observed in one male and one female in the main group and one male in the recovery group. It was considered to be little toxicological meaning or an individual difference since it showed low incidence. Stillbirth was observed in one female at 800 mg/kg/day. It was
considered to be a test substance-related adverse effect.

No clinical signs were observed in males and females of the main and recovery groups in the detailed examinations conducted once a week.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the main group, one female in the 0 mg/kg/day group (PPD 1) and 3 females in the 800 mg/kg/day group (GD 21, GD 22, GD 23) died. In the recovery group, one female in the 0 mg/kg/day group (Day 60) was found dead. Since there were no deaths at 800 mg/kg/day in the recovery group, the death was considered as test substance-related reproductive toxicity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the main group, significant low values in body weight were noted in females at 800 mg/kg/day on GD 14 and GD 20 compared to the controls. It was considered to be a test substance-related reproductive toxicity since there was no significant adverse effect on the body weight at 800 mg/kg/day in the recovery group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference in food consumption was noted in males and females of the main and the recovery groups when compared to the control group.
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 effect was observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because they were small differences and the values were within the range of historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effect was observed in any animal in the main and recovery groups. There was an exception of high BUN and Crea values. It was considered to be incidental since it showed low incidence (only one female) and no microscopic lesion in the kidney of the female. Other statistical significances were considered not to be test substance-related changes because they were small differences and/or the values were within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no effect was noted in urinalysis in males of any dosing group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on the auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group. In the main group, there was no test substance-related effect in the grip strength test when compared to the control group. In the recovery groups, a statistically significant increase in the hindlimb grip strength was noted in females at 800 mg/kg/day. However, it had little toxicological significance because it was a small difference. In the main and recovery groups, there was some statistical significance in the spontaneous motor activity. However, it had little toxicological relevance because it showed small difference, temporary change or no dose-dependency.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A significant decrease in T4 was noted in males at 800 mg/kg/day. However, it was considered to have little toxicological meaning since there was no abnormal microscopic finding in the thymus of males.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The relative organ weights of some organs (the brain, heart and kidney) were statistically increased in males at 800 mg/kg/day. However, these changes were not considered to be test substance-related adverse effects since they were related to low fasted body weight at 800 mg/kg/day. Other statistical significances in the absolute and/or relative organ weights were considered not to be test substance-related effects because they were small differences and/or the values were within the range of historical reference data.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dead animals
Three dams were found dead at 800 mg/kg/day just before or during parturition. Among the three dams, only one dam showed small thymus and spleen. There were no abnormal macroscopic findings in the other dams. In addition, two females the 0 mg/kg/day group were found dead. One dam died after dystocia and the other one female died during the recovery period. Macroscopic examination revealed multiple black foci in the glandular stomach and small thymus and spleen in one dam. However, no abnormal macroscopic findings were observed in the female of the recovery group.

Surviving animals (Terminal sacrifice):
Four dams whose pups were all dead showed small thymus (4/4) and spleen (2/4) at 800 mg/kg/day. Small thymus was also observed in two dams at 200 mg/kg/day. However, dams whose pups were survived at 800 mg/kg/day did not show any macroscopic changes. One non-delivered female in the 0 mg/kg/day group was revealed as non-pregnant animals, and there were no abnormal findings noted. A subcutaneous mass in the chest region was observed in one dam at 50 mg/kg/day. All other macroscopic findings showed a lack of dose-dependent relationship; were found at an isolated frequency, and were considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dead animals:
There were no microscopic findings related to the test substance. However, it was considered that the test substance affected the deaths at 800 mg/kg/day since only dams died at 800 mg/kg/day before or during parturition. Slight to severe atrophy of the lymphoid organs (thymus and spleen) was noted in 2 dead dams at 800 mg/kg/day, and slight/moderate adrenocortical hypertrophy and minimal to moderate serous atrophy of the bone marrow (femur and sternum) were noted in 3 dead dams at 800 mg/kg/day. These findings were considered to be stress-related secondarily to exacerbated/moribund condition. Moderate suppurative inflammation in the spleen was observed in one dead dam at 800 mg/kg/day. However, this finding was deemed to be incidental, and not to be primary cause of death based on the severity of the finding. In addition, acute centrilobular necrosis in the liver was noted in one dead dam at 800 mg/kg/day. This finding was deemed to be not associated with the test substance since the finding was of isolated frequency and could be occurred as ischemicevent during agonal phase. In one dead dam at 0 mg/kg/day, there were severe atrophy of the lymphoid organs (thymus and spleen), slight adrenocortical hypertrophy and minimal erosion in the glandular stomach. This dam was found dead after dystocia, thus it was considered that dystocia contributed to those stress-related findings and deaths. Another dead female which died during the recovery period did not show any abnormal findings, and the cause of death could be not determined.

Surviving animals (Terminal sacrifice):
Microscopic examination did not reveal any target organ in either terminal or recovery groups. Severe thymic atrophy was noted in 4 dams at 800 mg/kg/day, and moderate or severe atrophy of white pulp in the spleen was found in 2 dams at 800 mg/kg/day. While the atrophic changes of the lymphoid organs were consistently observed in dams whose pups were all dead or in a dam on stillbirth, those were not seen in other dams at 800 mg/kg/day. Based on the distribution of the incidence, these findings were considered to be secondary to poor condition/stress associated with the test substance. Minimal/slight thymic atrophy was noted in 2 dams at 200 mg/kg/day. However, the cortex of the thymus in pregnant rat can be slightly atrophied, and the dams showed normal condition and normal lymphocyte relative count (referring to biochemical data). Therefore, it was considered to be not of toxicological significance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dead animals:
There were no microscopic findings related to the test substance.

Surviving animals (Terminal sacrifice):
Microscopic examination did not reveal any target organ in either terminal or recovery groups. The subcutaneous mass observed in one dam at 50 mg/kg/day corresponded to mammary gland adenocarcinoma. The tumors was considered to be not related to the test substance since the mammary tumor was found at an isolated frequency at 50 mg/kg/day and can occur spontaneously in female rats of this breed and age.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle lengths (day) of females in the 0 (control), 50, 200 and 800 mg/kg/day dosing groups were 4.2, 4.1, 4.1 and 4.0 days, respectively. There was no statistically significant difference in any dosing group. The estrous cycle of all females including the non-pregnant female on the day of necropsy was noted as diestrus.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In the control group and 50, 200 and 800 mg/kg/day dosing groups, the mating periods were 2.9, 2.2, 2.2 and 2.6 days, the mating indices were all 100.0%, the gestation periods were 22.2, 22.3, 22.2 and 22.4 days, and the fertility indices of animals of both sexes were 91.7, 100.0, 100.0 and 100.0%, respectively. There was no statistically significant difference in any dosing group. In the control group and 50, 200 and 800 mg/kg/day dose groups, the gestation indices were 100.0, 100.0, 100.0 and 75.0%, the post-implantation loss rates were 17.1, 8.4, 11.9 and 65.7%, the live birth indices were 82.9, 91.7, 88.1 and 45.8%, the mean litter sizes were 14.5, 14.2, 12.1 and 11.3%, the viability indices on Postnatal Day (PND) 0 were 90.2, 99.0, 100.0 and 50.9, and the viability indices on PND 4 were 99.2, 94.3, 98.8 and 71.7%, respectively. In the 800 mg/kg/day dose group, high post-implantation loss and low viability of pups on PND 4 were noted. Furthermore, decreases of live birth index and viability of pups on PND 0 were not statistically significant, but they were of toxicological relevance since the differences were substantial. In addition, abnormal delivery was observed in one female at 0 mg/kg/day (dystocia) and 3 females at 800 mg/kg/day (stillbirth or dystocia). The sex ratios on PND 0 were 1.3, 0.9, 1.4 and 0.8, respectively.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male
Remarks on result:
other: No adverse effects observed.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
body weight and weight gain
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The viability indices on Postnatal Day (PND) 0 were 90.2, 99.0, 100.0 and 50.9, and the viability indices on PND 4 were 99.2, 94.3, 98.8 and 71.7%, respectively. In the 800 mg/kg/day dose group, low viability of pups on PND 4 were noted. Furthermore, decreases of viability of pups on PND 0 were not statistically significant, but they were of toxicological relevance since the differences were substantial.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test substance-related changes in the body weights of male or female pups at 0, 50, 200 and 800 mg/kg/day on PNDs 0, 4 and 13.
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
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic examination was conducted on the thyroids of pups due to the decreases in serum thyroxine levels in pups. However, there were no abnormal morphological changes in the thyroids of pups.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance:
There were no test substance-related changes in the AGD indexes of male and female pups at 0, 50, 200 and 800 mg/kg/day on PND 4.

Nipple retention:
The nipple number was 0 in male pups at 0, 50, 200 and 800 mg/kg/day on PND 12. There was no nipple retention in male pups at 50, 200 and 800 mg/kg/day on PND 12.

Thyroid Hormone Analysis:
A significant decrease in T4 was noted in male pups at 800 mg/kg/day. However, it was considered to have little toxicological meaning since there was no abnormal microscopic finding in the thymus of male pups.

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:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive toxicity was considered to be 800 mg/kg/day for males and 200 mg/kg/day for females. The NOAEL for developmental toxicity was considered to be 200 mg/kg/day for pups.
Executive summary:

A combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening test was performed to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic toxicity and reproduction/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential of the test substance when administered orally to male and female rats at dose levels of 0 (control), 50, 200 and 800 mg/kg/day. All males of the main and recovery groups survived the duration of the study. One dam in the main group and one female in the recovery group were found dead at 0 mg/kg/day. Three pregnant females of the main group were found dead at 800 mg/kg/day before or during parturition. Four dams whose pups were all dead were observed at 800 mg/kg/day. Stillbirth was observed in one female at 800 mg/kg/day. Low value in the body weight was noted in pregnant females at 800 mg/kg/day in the main groups. Atrophy of the lymphoid organs, adrenocortical hypertrophy and/or serous atrophy of the bone marrow were noted in three dead dams. These findings were considered to be stress-related secondarily to exacerbated/moribund condition. Thymic atrophy and/or atrophy of white pulp in the spleen were observed in dams whose pups were all dead at 800 mg/kg/day. An increase in post-implantation loss rate and decreases in birth index and viability index of pups on PNDs 0 and 4 were noted at 800 mg/kg/day. Abnormal delivery was observed in one female of the control group and three females at 800 mg/kg/day. In conclusion, the NOAEL for reproductive toxicity was considered to be 800 mg/kg/day for males and 200 mg/kg/day for females. The NOAEL for developmental toxicity was considered to be 200 mg/kg/day for pups.