Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2015 to 24 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

various with no impact on results or integrity of study (see attachement)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Appearance/physical state: Dark black solid
Storage conditions: Room temperature, protected from light, under inert gas

Test animals

Species:

rat

Strain:

other: CD [Crl:CD(SD)

Details on test animals or test system and environmental conditions:

ANIMAL ACQUISITION AND ACCLIMATION
- A total of 60 males (for breeding purposes only) and 124 female CD [Crl:CD®(SD)] rats (approximately 10 weeks of age) were received from Charles River Laboratories, Raleigh, North Carolina, on February 23, 2015.
- The animals were acclimated for two weeks prior to pairing for mating. During the acclimation period, the animals were observed twice daily with respect to general health and any signs of disease.
- Following the acclimation period, one male was housed with one female in the cage of the male. Positive evidence of copulation was established by daily inspection for a copulatory plug in situ or vaginal lavage
for sperm. The day on which positive evidence of copulation was observed was considered GD 0.
- All male animals had body weights recorded upon arrival. Data for the male animals are not reported but are maintained in the study file.
- All female animals were given a detailed clinical examination prior to selection, and body weights were recorded upon arrival and prior to selection and dosing on GD 0.
- The Study Director reviewed the GD 0 body weight and detailed observation data for all female animals and gave final approval for assignment to study.
- The findings from the pretest clinical examinations recorded during the acclimation period and the body weights recorded upon arrival are not reported but are maintained in the study file.
- One female animal was excluded from study upon receipt due to being found dead in the shipping container.

RANDOMIZATION, ASSIGNMENT TO STUDY AND MAINTENANCE
- Using consecutive placement in a block design, 100 female animals (weighing 232 to 300 g, at randomization) were assigned to the control and treatment groups identified in the group assignments table (below).
- The order in which they were assigned corresponded to the day on which positive evidence of mating was observed.
- Extra animals obtained, but not placed on study, were euthanized via carbon dioxide inhalation. Euthanasia was confirmed via exsanguination of the abdominal vena cava and the carcasses were discarded.
- Each animal was assigned an animal number to be used in the Provantis data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.
- The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room, except during pairing. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day. Temperature and humidity were monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79 °F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported but are maintained in the study file.
- Block Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water
analyses are retained in the Archives. The Study Director was not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

VEHICLE AND TEST ARTICLE PREPARATION
- Fresh vehicle, peanut oil, was prepared for use on study weekly by bubbling (sparging) nitrogen though the oil for at least 15 minutes. The vehicle was stored refrigerated at 2 to 8 °C under a blanket of nitrogen.
- The test article was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations.
- The test article was mixed with the peanut oil to achieve the desired concentrations. Based on lower than expected recovery rates and variability in the homogeneity results for the Week 1 preparations, the formulation
was re-prepared for dosing during Week 1. For the Week 1 re-preparation and each subsequent preparation, the test article was ground with a mortar and pestle prior to weighing.
- Formulations of the test article were prepared weekly at nominal concentrations of 5, 12.5, and 25 mg/mL, and were stored refrigerated at 2 to 8 °C under a blanket of nitrogen to minimize contact with air.

JUSTIFICATION FOR ROUTE OF EXPOSURE
- The oral route is a potential route of accidental exposure of this test article in humans.

JUSTIFICATION OF DOSE LEVELS
- The dose levels were selected by the Sponsor on the basis of data from previous studies.
- In a rat 28-day study, animals were dosed at 100/60/50, 30, and 10 mg/kg/day. The high-dose level was found to have excessive toxicity at 100 mg/kg/day (decreases in body weight and food consumption, as well as hunched posture) and was therefore lowered to 50 mg/kg/day. In the females at 30 mg/kg/day, there was a slight decrease in body weight during the first week of dosing. Animals at 10 mg/kg/day were comparable to controls.
- Based on these findings, a high-dose level of 50 mg/kg/day was selected for the prenatal development toxicity study to show some morbidity and a low-dose level of 10 mg/kg/day was selected to show no adverse effects. A mid-dose level of 25 mg/kg/day was selected to provide an intermediate dose.

ADMINISTRATION
- The vehicle and test article were administered once per day from GD 0 to 19 at approximately the same time of day (± 2 hours from the GD 0 dose) via oral gavage.
- The dose levels for the treated groups were 10, 25, and 50 mg/kg/day at a dose volume of 2 mL/kg.
- The control group received the vehicle in the same manner and at the same volume as the treated groups.
- The vehicle and test article were stirred at room temperature for at least 30 minutes prior to dosing and remained stirring throughout dosing.
- Individual doses were based on the most recent body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSING FORMULATIONS
- Dosing formulations prepared for the study were evaluated for homogeneity and concentration.
- Appropriate samples (see attached table) were collected using a positive displacement pipette, while stirring and placed into amber glass scintillation vials.
- Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples were discarded.
- Stability was established for 10 days at refrigerated temperature (2 to 8°C) and purged with nitrogen under MPI Research, Inc., Study Number 1928-015.

ANALYSES
- All analytical work was conducted by MPI Research, Inc., using an analytical method developed and validated under MPI Research, Inc., Study Number 1928-015 (see Appendix B, attached).

Details on mating procedure:

Refer to details of mating given in Appendix H (attached)

Duration of treatment / exposure:

Gestation Day 0 to 19

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 mated females

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGESIDE OBSERVATIONS
- All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.
- On occasion, veterinary consultations were conducted during the course of the study.
- All treatments and observations were recorded.
- The medical treatments and observations are not reported but are maintained in the study file.

DETAILED CLINICAL OBSERVATIONS
- Daily from GD 0 through 20 (60 to 90 minutes post-dose on dosing days), each animal was removed from the cage and given a detailed clinical examination.
- On occasion, clinical observations were recorded at unscheduled intervals.
- The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

BODY WEIGHT AND BODY WEIGHT CHANGES
- Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20.
- Individual body weight change was calculated for the following GD intervals: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, and 0-20.
- Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.

FOOD CONSUMPTION
- Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals.

MATERNAL NECROPSY
- A complete necropsy was performed on all dams under procedures approved by a veterinary pathologist.
- Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy.
- The presence of lesions or other abnormal conditions in the dam were noted and described in the study records.

Ovaries and uterine content:

OVARIAN AND UTERINE EXAMINATION
- On GD 20, each female was euthanized by carbon dioxide inhalation, followed by exsanguination of the abdominal vena cava and immediately subjected to a cesarean section. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded.
- Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and
late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
- Each implant was categorized according to the following criteria. Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics. The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
- Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. The foci, if detected, were considered early resorptions, and data from this female were included in mean calculations. If no foci were detected, the female was considered to be nonpregnant.
- Maternal necropsies and subsequent fetal evaluations were conducted without knowledge of the treatment groups in order to minimize bias.

Fetal examinations:

FETAL EXAMINATIONS
- Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations.
- Fetuses were then euthanized by intraperitoneal injection of euthanasia solution.
- Approximately one-half of the fetuses in each litter were placed in Bouin’s solution and the remaining fetuses were fixed in alcohol.
- All fetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique.
- The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination.
- Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist.
- On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data.
- Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the fetuses. These artifacts are not reported, but are maintained in the study data.

Statistics:

STATISTICS
- Comparisons were made between the control group (1) and treatment groups (2, 3 and 4).
- The endpoints that were analyzed and the methods of analysis that were employed are presented in the attached table.
- Further description of statistical methods is given below.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Detailed clinical observations are summarized in Table 1 (attached).
- No effect of test item at 10 mg/kg/day was observed from the detailed post-dose clinical examinations. Clinical findings observed in this group occurred at low incidence or with similar frequency as in controls and were considered unrelated to the test article.
- Salivation was observed at least once in 4/25 (16%) and 6/25 (24%) animals in the 25 and 50 mg/kg/day dose groups, respectively. In the absence of salivation among animals in the control or 10 mg/kg/day dose group, its occurrence in these groups was considered test article-related.
- In the 50 mg/kg/day group, 19/25 animals (76%) were observed on one or more occasions as appearing thin and 17 of these animals were either not pregnant or experienced a failed pregnancy (i.e., uterine implants comprised entirely of resorbing fetuses or pregnancy confirmed solely on the basis of stained uterine foci at GD 20).
- An increased incidence of brown or red discharge from the anus or vulva was also observed in the 50 mg/kg/day group. One or both of these findings were observed in 11/25 animals (44%) in this group and seven of these animals experienced a failed pregnancy.
- Other clinical findings observed among the 25 and 50 mg/kg/day groups occurred at low incidence or with similar incidence as in controls and were considered unrelated to the test article.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

- All animals in the control and treated groups survived to scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Maternal body weight and body weight change data (during gestation) are summarized in Tables 2 and 3, respectively (attached).
- No effect of test item at 10 mg/kg/day was observed on gestation body weights or body weight change. In the 25 mg/kg/day group, slight although statistically significant differences in gestation body weights relative to controls were observed on GD 3 (-5%), GD 6 (-7%), GD 9 (-4%) and GD 20 (-6%), and statistically significant differences in weight change were observed as a weight loss GD 0 to 3 (-5.9 g vs. a weight gain of 12.9 g in controls) and lower weight gains GD 18 to 20 (-46%) and GD 0 to 20 (-23%).
- In the 50 mg/kg/day group, body weights differed statistically from controls throughout gestation ranging from 6% to 30 % lower and weight losses over GD 0 to 3 (-11.9 g), GD 3 to 6 (-9.6 g) and GD 6 to 9 (-3.5 g) and lower weight gains for the remainder of gestation ranging from 56% to 94% relative to controls.
- These effects on gestation weights and weight change in the 25 and 50 mg/kg/day dose groups were considered test article related, correlated with low food consumption, and in the 50 mg/kg/day animals consistent with an increased incidence of failed pregnancies.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Maternal food consumption (during gestation) is summarized in Table 4 (attached).
- No adverse effect of test item at 10 mg/kg/day was observed on gestation food consumption. Mean food consumption in this group over GD 0 to 3 at 15.8 g/animal/day differed statistically from controls (18.3 g) but the effect was transient and not considered adverse as food consumption for the remainder of gestation was comparable to controls.
- In the 25 mg/kg/day group, mean food consumption was statistically lower relative to controls over GD 0 to 3 (-52%), GD 3 to 6 (-39%), GD 18 to 20 (-29%), and GD 0 to 20 (-21 %). In the 50 mg/kg/day group, food consumption was statistically lower relative to controls throughout gestation with differences ranging from -61% to -72%.
- The lower food consumption in the 25 and 50 mg/kg/day groups was considered test article related correlating with effects observed on gestation weights and weight change in these groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- No effect of test item at 10 and 25 mg/kg/day was observed from the maternal macroscopic examinations. The few findings observed among these animals occurred at low incidence and considered unrelated to the test article.
- In the 50 mg/kg/day group, small thymus was observed in 4/25 (16 %) animals. This was not observed among the control or animals in the 10 or 25 mg/kg/day groups and its increased occurrence in the 50 mg/kg/day group was considered test article related.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- Corpora lutea and uterine examination data are summarized in Table 5 (attached).
- Gravid uterine weights and adjusted body weight/body weight changes are summarized in Table 6 (attached).
- No effect of test item at 10 and 25 mg/kg/day was observed on corpora lutea counts and uterine implantation data. The mean number of corpora lutea, implantation sites, viable fetuses, litter size and resorption sites (early, late and total [early plus late]) per dam and mean preimplantation and postimplantation loss in these groups were comparable to controls.
- In the 50 mg/kg/day group, the mean number of implantation sites, viable fetuses, and litter size were statistically lower than controls and the mean postimplantation loss and mean number of resorption sites (early, late and total) per dam were statistically higher than controls. The mean number of corpora lutea and mean preimplantation loss in the 50 mg/kg/day group were comparable to controls.
- The increased postimplantation loss observed in the 50 mg/kg/day dose group was considered test article related.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

- Of the 17 pregnancies in the 50 mg/kg/day group, 10 (59 %) were comprised of all resorbing fetuses (100 % postimplantation loss) three of which were identified only after staining of the uterus.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

- Pregnancy index was 92 %, 92 %, 100 % and 68 % in the control, 10, 25 and 50 mg/kg/day dose groups, respectively. There were two nonpregnant females in each of the control and 10 mg/kg/day groups and 8 nonpregnant in the 50 mg/kg/day group.
- The low pregnancy index in the 50 mg/kg/day dose group was suggestive of toxicity to the pre-implanted fertilized ovum particularly in consideration of the high pregnancy rates in the other study groups which used these same males for mating.
- Overall, there were 23, 23, 25 and 7 females with litters containing viable fetuses for evaluation on GD 20 in the control, 10, 25 and 50 mg/kg/day groups, respectively.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

MEAN GRAVID UTERINE WEIGHTS
- Mean gravid uterine weights, adjusted GD 20 body weights, and adjusted body weight change GD 0 to 20 in the 10 mg/kg/day dose group were comparable to controls.
- In the 25 mg/kg/day group, mean gravid uterine weight was comparable to controls but the adjusted weight and adjusted body weight change were statistically lower. In the 50 mg/kg/day group, gravid uterine weight, adjusted body weight and adjusted body weight change were statistically lower than controls.
- These effects on adjusted body weights and adjusted weight change in the 25 and 50 mg/kg/day dose groups were considered test article related and consistent with effects observed at these dose levels on actual GD 20 gestation weights and weight change GD 0 to 20.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: thymus

Results (fetuses)

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Fetal body weight are summarized in Table 7 (attached).
- No effect of test item at 10 and 25 mg/kg/day was observed on fetal body weights.
- In the 50 mg/kg/day group, mean fetal weights for the combined sexes were 28% lower than controls, 26% lower for male fetuses, and 29% lower for female fetuses. These differences were statistically significant and considered test article-related. However, it should be noted that mean fetal weights (distinguished by sex and for the combined sexes) in the control and treated groups were outside the low range of recent historical control data for the laboratory.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- Fetal sex ratios are summarized in Table 5 (attached).
- No effect of test item at the dose levels evaluated was observed on fetal sex ratios.
- These ratios ranged from 40.0% to 48.3% in the treated groups and were comparable to the 50.5% in controls.

External malformations:

no effects observed

Description (incidence and severity):

- Individual fetal external observations are summarized in Table 8 (attached) and the overall incidence of fetal external malformations and variations is summarized in Table 11 (attached).
- No effect of test item at the dose levels evaluated was observed from the fetal external examinations.
- The few external malformations observed among the 10 and 25 mg/kg/day fetuses occurred at low incidence (single fetus affected), were dissimilar in nature, and considered spontaneous in nature and unrelated to the test article.
- No external malformations were observed in fetuses from the 50 mg/kg/day group.
- In the control group, one fetus had a cranial malformation (exencephaly).
- Likewise, no effect of the test article was observed from developmental variations observed at the fetal external examinations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

- Individual fetal skeletal observations are summarized in Table 10 (attached) and the overall incidence of fetal skeletal malformations and variations is summarized in Table 13 (attached).
- No effect of test item was observed at the dose levels evaluated from fetal skeletal malformations. In the 25 mg/kg/day group, fusion of the rib costal cartilage was observed in two fetuses in a single litter. This has a low historical occurrence in this laboratory and its low incidence in in this group was considered spontaneous in nature and unrelated to the test article.
- No skeletal malformations were observed in the 10 or 50 mg/kg/day fetuses.
- In the control group, malformations involving misshapen cranial bones were observed in the fetus noted at external examination with a cranial malformation (exencephaly).
- Skeletal variations observed in the 10 mg/kg/day dose group occurred with comparable incidence as controls and no effect of test article was observed in these data. In the 25 mg/kg/day group, statistically significant increases relative to controls were observed in the litter incidences of the skeletal variations “cervical vertebral neural arches – additional ossification centers” (36% vs. 8.7% in controls) and “rudimentary rib structures” (68% vs.
21.7% in controls). In the 50 mg/kg/day group, the litter incidences of “cervical vertebral neural arches - additional ossification centers” at 71.4% and “interparietal bone of the skull incompletely ossified” at 71.4% differed statistically from control incidences of 8.7% and 4.3%, respectively.
- The litter incidences of these skeletal variations in the 25 and 50 mg/kg/day groups were also outside the range of recent historical control data for the laboratory.
- Since litter incidences of other skeletal variations in the 25 and 50 mg/kg/day dose groups were comparable to controls and generally within the range of recent historical control for the laboratory, their increased occurrence in this study was considered fortuitous and unrelated to the test article.

Visceral malformations:

no effects observed

Description (incidence and severity):

- Individual fetal visceral observations are summarized in Table 9 (attached) and the overall incidence of fetal visceral malformations and variations is summarized in Table 12 (attached).
- No effect of test item was observed at the dose levels evaluated from the fetal visceral examinations.
- The few visceral malformations observed among fetuses in the treated groups occurred at low incidence affecting single fetuses, were dissimilar in nature, and considered spontaneous in nature and unrelated to the test article.
- No visceral malformations were observed in fetuses from the control group.
- Likewise, no effect of the test article was observed from developmental variations observed at the fetal visceral examinations.
- The few variations observed among the treated fetuses occurred at low incidence or were comparable to controls and were considered unrelated to the test article.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: maternal toxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 10 mg/kg/day and the NOAEL for developmental toxicity was 25 mg/kg/day. The test item was not teratogenic in the rat at the dose levels evaluated.

Executive summary:

GUIDELINE

A prenatal developmental toxicity study was conducted to evaluate the possible adverse effects of the test article following repeated exposure to pregnant rats. The study was based on the Guideline 414, Prenatal Developmental Toxicity Study, the Organization of Economic Cooperation and Development (OECD) Guideline for Testing of Chemicals, adopted January 22, 2001; the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011; and the U.S. Department of Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3).

 

METHODS

The evaluation included systemic toxicity, female reproductive performance, and evaluation of the fetuses. The vehicle, arachis oil NF (peanut oil), or test article was administered to mated CD [Crl:CD(SD)] rats once daily via oral gavage from Gestation Day (GD) 0 through 19. Mated females were assigned to dose levels of 0, 10, 25 or 50 mg/kg/day. Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, or skeletal examination. Homogeneity of the dosing formulations was confirmed at the low- and high-concentration levels in Week 1. Average concentrations of test item in Weeks 1 and 4 ranged between 87.5 % and 100.1 % of nominal with percent RSDs ranging between 0.675 and 10.107, confirming that animals were receiving the appropriate dose levels. No test article was found in control samples.

 

RESULTS

All animals survived to schedule termination on presumed GD 20. At 10 mg/kg/day, no effect of the test article was observed on clinical findings, gestation body weights, body weight change, pregnancy rates, uterine implantation data, fetal sex ratios, fetal body weights or fetal external, visceral or skeletal examinations. A slight although statistically significant decrease in food consumption over GD 0 to 3 in this group was transient and not considered adverse. At 25 mg/kg/day, test article-related effects were observed from clinical findings (salivation), low gestation body weights, low gestation body weight gain and low food consumption. No effects at this dose level were observed on pregnancy rate, uterine implantation data, fetal sex ratios, fetal body weights or fetal external,

visceral or skeletal examinations. At 50 mg/kg/day, toxicity to the pre-implanted embryo and fetus was observed from the low pregnancy rate (68%) in this group and increased incidence of failed pregnancies (i.e., uterine implants comprised entirely of resorbing fetuses or pregnancy confirmed solely on the basis of stained uterine foci at GD 20). Of the 17 pregnancies in this group (8 females were not pregnant), 10 females failed to retain fetuses to GD 20. The high number of failed pregnancies contributed to the increased occurrence of red/brown discharge from the anus and/or vulva and thin appearance observed clinically in this group, and lower gestation body weights, lower body weight change and lower food consumption. Other effects observed at 50 mg/kg/day were increased postimplantation loss, decrease in number of fetuses, increase in resorption sites, lower fetal body weights and at maternal necropsy an increased occurrence of small thymus (4/25 animals affected [16%]). No effects at 50 mg/kg/day were observed on fetal sex ratios or fetal external, visceral or skeletal evaluations.

 

CONCLUSION

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 10 mg/kg/day and the NOAEL for developmental toxicity was 25 mg/kg/day. The test item was not teratogenic in the rat at the dose levels evaluated.