1,3-diisopropylbenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diisopropylbenzene

EC Number:

202-773-1

EC Name:

1,3-diisopropylbenzene

Cas Number:

99-62-7

Molecular formula:

C12H18

IUPAC Name:

1,3-bis(propan-2-yl)benzene

Test material form:

liquid

Details on test material:

The test item was 1,3-diisopropylbenzene (DIPB or m-DIPB). Test item information is presented below:

IDENTIFICATION: 1,3-diisopropylbenzene; Lot no. 6F50423000 [WIL ID no. 150134]
PHYSICAL DESCRIPTION: Clear, colorless liquid
DATE OF RECEIPT: 04-May-2015

A Certificate of Analysis for the test item was provided by the Sponsor. The purity of the test item was 97.4%. The test item was stored at room temperature, and was considered stable under this condition. A reserve sample of the test item was collected and stored in the WIL Research Archives.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, on 21-May-2015.
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are fully accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
- Diet: ad libitum, the basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Water: ad libitum, municipal water supplying delivered by an automatic watering system
- Acclimation period: a minimum of 12 days for acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3°C to 22.3°C
- Humidity (%): 46.0% to 57.5%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

The control item was prepared approximately weekly for administration to the control group (Group 1); aliquots were prepared for daily dispensation to the control group and stored at room temperature.

GroupNumber Treatment Dosage Level (mg/kg/day) Dosage Volume (mL/kg)
1 CONTROL 0 1.16
2 m-DIPB 100 0.12
3 m-DIPB 300 0.35
4 m-DIPB 1000 1.16

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dosage levels were selected based on the results of a previous 28-day study. Analyses to demonstrate the homogeneity, stability, and concentration of the test item were not conducted in this study. The test item was expected to be stable for the duration of testing. Characterization of the test item was conducted previously and the test item was applied in pure form.

Details on mating procedure:

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated. The experimental design consisted of 3 test item-treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony. Body weight values ranged from 220 g to 284 g on gestation day 0.

Duration of treatment / exposure:

19 days

Frequency of treatment:

once daily

Duration of test:

19 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

Timed-pregnant female rats were treated with the test article by oral gavage from gestation day 6 to 19, at which point a cessarian section was performed and data on the dams and fetuses was obtained.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Individual maternal body weights were recorded on gestation days 0-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 1-3, 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” on the individual report tables. Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 0-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Ovaries and uterine content:

UNSCHEDULED DEATHS
A gross necropsy was performed on females that were found dead during the course of the study. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally. The females and all products of conception were discarded.

GESTATION DAY 20 LAPAROHYSTERECTOMY
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss
(Salewski, 1964). The kidney, liver, and stomach from all females euthanized at the scheduled necropsy were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Other maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Fetal examinations:

FETAL MORPHOLOGICAL EXAMINATION
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976) as described in the protocol. Fetuses were then examined for skeletal malformations and developmental variations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the
means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20 with 13 dead fetuses (with no apparent malformations) in utero; this female was noted with rales on gestation day 2 but had no remarkable clinical findings between gestation day 2 through the day of death. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 (pregnant) was found dead on gestation day 12 following intermittent body weight losses during gestation day 1-11 (9.2% body weight loss). During gestation day 10-11, this animal consumed only 1 g of feed (with corresponding decreased defecation). At necropsy, this female had lungs that were not fully collapsed, suggesting that the death was likely related to the dosing procedure, and therefore was not considered test item-related. Female no. 8129 (pregnant) was found dead on gestation day 12; at necropsy, this animal had lungs that were not fully collapsed (with dark red areas) and foamy contents in the trachea. The death of female no. 8129 was likely related to the dosing procedure, and therefore was not considered test item-related. All other females survived to the scheduled necropsy.
Increased incidences of clear and/or red material around the mouth were noted at 1 hour following dose administration for the surviving 1000 mg/kg/day group females when compared to the control group. These findings were noted as early as gestation day 1 and persisted through gestation day 19. Additionally, a slight increase of these same observations were noted sporadically in the 300 mg/kg day group females when compared to the control group. There were no test item-related clinical observations noted in the 100 mg/kg/day group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Due to a significant (p<0.01) mean maternal body weight loss in the 1000 mg/kg/day group following the first day of treatment (gestation day 1-2), mean maternal body weight gain in this group during gestation days 1-3 was significantly (p<0.01) lower than the control group value (1 g compared to 10 g in the control group). Mean body weight gains in the 1000 mg/kg/day group were similar to the control group values during gestation days 3-6, 6-9, 9-12, and 12-15. Significantly (p<0.05) lower mean body weight gains compared to the control group were noted in the 1000 mg/kg/day group during gestation days 15-20 and when the overall treatment period (gestation days 1-20; 13.1% lower) was evaluated. As a result, mean body weights in the 1000 mg/kg/day group during gestation days 18-20 were slightly lower (up to 5.3%) than the control group values; the differences for gestation days 18 and 19 were significant (p<0.05).
Although not statistically significant, mean net body weight gain (12.5%) in this group were lower compared to the control group. The aforementioned changes were considered test item-related. The lower mean net body weight gain in the 1000 mg/kg/day group was not of sufficient magnitude to result in a lower mean net body weight. As a result of the lower mean number of viable fetuses in this group, gravid uterine weight was 12.2% lower than in the control group. Because the lower number of viable fetuses was not considered to be test item-related, the lower gravid uterine weight was not considered to be test item-related.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 300 mg/kg/day groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was lower than the control group values during gestation days 1-3; the differences were significant (p<0.01). The lower food consumption values
in this group during the first 3 days of treatment corresponded to lower body weight gains, and were therefore also considered test item-related. With the exception of slightly higher mean g/kg/day food consumption values during gestation days 9-12 and 15-20 (significant at p<0.05), mean food consumption values in the 1000 mg/kg/day group were similar to the control group values throughout the remainder of the treatment period (gestation days 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20). Test item-related significantly (p<0.05 or p<0.01) lower mean food consumption was noted in the 100 and 300 mg/kg/day groups during gestation days 1-3. In the absence of an effect on body weights and body weight gains in these groups, the initial food consumption decrements were considered nonadverse. Mean food consumption in these groups was generally similar to the control group for the remainder of the treatment period, with the following exception, higher mean food consumption was noted during gestation days 12-15 (g/animal/day and g/kg/ day) and 15-20 (g/kg/day only) in the 300 mg/kg/day group; differences from the control group were significant (p<0.01 or p<0.05). The aforementioned higher mean food consumption was not attributed to test item administration because the direction of change was not considered toxicologically relevant.

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

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20; at necropsy, this animal had 13 dead fetuses (with no apparent malformations) in utero. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 was found dead on gestation day 12; this animal had lungs that were not fully collapsed and 12 normally developing implantations in utero. Female no. 8129 (also found dead on gestation day 12) had lungs that were not fully collapsed (with dark red areas), foamy contents in the trachea, and 16 normally developing implantations in utero. All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 100, 300, and 1000 mg/kg/day. Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

None found

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

None found

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was not a significant change in early or late resorptions

Dead fetuses:

no effects observed

Description (incidence and severity):

None

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not a valid metric of the study

Changes in number of pregnant:

not examined

Description (incidence and severity):

Not a valid metric of the study

Other effects:

not specified

Details on maternal toxic effects:

MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20 with 13 dead fetuses (with no apparent malformations) in utero; this female was noted with rales on gestation day 2 but had no remarkable clinical findings between gestation day 2 through the day of death. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 (pregnant) was found dead on gestation day 12 following intermittent body weight losses during gestation day 1-11 (9.2% body weight loss). During gestation day 10-11, this animal consumed only 1 g of feed (with corresponding decreased defecation). At necropsy, this female had lungs that were not fully collapsed, suggesting that the death was likely related to the dosing procedure, and therefore was not considered test item-related. Female no. 8129 (pregnant) was found dead on gestation day 12; at necropsy, this animal had lungs that were not fully collapsed
(with dark red areas) and foamy contents in the trachea. The death of female no. 8129 was likely related to the dosing procedure, and therefore was not considered test item-related. All other females survived to the scheduled necropsy. Increased incidences of clear and/or red material around the mouth were noted at 1 hour following dose administration for the surviving 1000 mg/kg/day group females when compared to the control group. These findings were noted as early as gestation day 1 and persisted through gestation day 19. Additionally, a slight increase of these same observations were noted sporadically in the 300 mg/kg day group females when compared to the control group. There were no test item-related clinical observations noted in the 100 mg/kg/day group.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Due to a significant (p<0.01) mean maternal body weight loss in the 1000 mg/kg/day group following the first day of treatment (gestation day 1-2), mean maternal body weight gain in this group during gestation days 1-3 was significantly (p<0.01) lower than the control group value (1 g compared to 10 g in the control group). Mean body weight gains in the 1000 mg/kg/day group were similar to the control group values during gestation days 3-6, 6-9, 9-12, and 12-15. Significantly (p<0.05) lower mean body weight gains compared to the control group were noted in the 1000 mg/kg/day group during gestation days 15-20 and when the overall treatment period (gestation days 1-20; 13.1% lower) was evaluated. As a result, mean body weights in the 1000 mg/kg/day group during gestation days 18-20 were slightly lower (up to 5.3%) than the control group values; the differences for gestation days 18 and 19 were significant (p<0.05). Although not statistically significant, mean net body weight gain (12.5%) in this group were lower compared to the control group. The aforementioned changes were considered test item-related. The lower mean net body weight gain in the 1000 mg/kg/day group was not of sufficient magnitude to result in a lower mean net body weight. As a result of the lower mean number of viable fetuses (see Section 6.5.) in this group, gravid uterine weight was 12.2% lower than in the control group. Because the lower number of viable fetuses was not considered to be test item-related, the lower gravid uterine weight was not considered to be test item-related. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 300 mg/kg/day groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

MATERNAL FOOD CONSUMPTION
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was lower than the control group values during gestation days 1-3; the differences were significant (p<0.01). The lower food consumption values in this group during the first 3 days of treatment corresponded to lower body weight gains, and were therefore also considered test item-related. With the exception of slightly higher mean g/kg/day food consumption values during gestation days 9-12 and 15-20 (significant at p<0.05), mean food consumption values in the 1000 mg/kg/day group were similar to the control group values throughout the remainder of the treatment period (gestation days 3-6, 6-9, 9-12, 12-15, 15-20, and 1-20). Test item-related significantly (p<0.05 or p<0.01) lower mean food consumption was noted in the 100 and 300 mg/kg/day groups during gestation days 1-3. In the absence of an effect on body weights and body weight gains in these groups, the initial food consumption decrements were considered nonadverse. Mean food consumption in these groups was generally similar to the control group for the remainder of the treatment period, with the following exception, higher mean food consumption was noted during gestation days 12-15 (g/animal/day and g/kg/ day) and 15-20 (g/kg/day only) in the 300 mg/kg/day group; differences from the control group were significant (p<0.01 or p<0.05). The aforementioned higher mean food consumption was not attributed to test item administration because the direction of change was not considered toxicologically
relevant.

MATERNAL NECROPSY DATA
Three females in the 1000 mg/kg/day group were found dead prior to the scheduled necropsy. Female no. 8085 was found dead on gestation day 20; at necropsy, this animal had 13 dead fetuses (with no apparent malformations) in utero. At necropsy, a cause of death was not determined but a relationship to the test item cannot be ruled out. Female no. 8089 was found dead on gestation day 12; this animal had lungs that were not fully collapsed and 12 normally developing implantations in utero. Female no. 8129 (also found dead on gestation day 12) had lungs that were not fully collapsed (with dark red areas), foamy contents in the trachea, and 16 normally developing implantations in utero. All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 100, 300, and 1000 mg/kg/day. Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group (3.8, 3.6, and 3.7 g, respectively) were slightly lower than the concurrent control group values (4.0, 3.8, and 3.9 g, respectively); the difference for females was significant (p<0.05). However, all high-dosage group values were equal to the respective mean values in the WIL Research historical control data; therefore, the slightly lower mean fetal body weights in the 1000 mg/kg/day group were not considered test item-related.
Mean fetal body weights in the 100 and 300 mg/kg/day groups were similar to the control group values; none of the differences were statistically significant. Intrauterine growth and survival was unaffected by test item administration at dosage levels of 100 and 300 mg/kg/day.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No changes found

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Although the mean litter proportion of pre-implantation loss was unaffected by test item administration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item administration.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not a valid metric for this study design

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were noted in 1 fetus in the 100 mg/kg/day group. Fetus no. 8092-05 in the 100 mg/kg/day group was observed with macroglossia and a palate with a high arch. Based on the occurrence in a single fetus and the absence of a
dose-response relationship, these malformations were not considered test item-related. No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in 2(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fetus no. 8074-09 in the 1000 mg/kg/day group (previously noted with visceral malformations) had malaligned sternebrae (nos. 2 and 3 [moderate] and 4 and 5 [severe]). Fetus nos. 8086-11 and 8086-13 in the control group and no. 8152-05 in the 100 mg/kg/day group had costal cartilage anomalies (fused, bifurcated, and/or malpositioned costal cartilages). The skeletal malformations noted in the 100 and 1000 mg/kg/day groups were not attributed to the test item because they occurred in single fetuses and/or similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. No test item-related skeletal developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data for definitive studies

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were noted in 3(2), 0(0), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Interventricular septal defects (≤ 2-mm in diameter opening in the anterior or posterior portion of the septum) were noted for fetus nos. 8086-04 and 8114-01 in the control group and fetus no. 8074-09 in the 1000 mg/kg/day group. Fetus no. 8074-09 in the 1000 mg/kg/day group also had a malpositioned atrium (the left atrium was located more dorsal than normal). The occurrences of interventricular septal defect and malpositioned atrium in the 1000 mg/kg/day group were not attributed to the test item because they occurred in a single fetus and/or similarly in the concurrent control group and the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group. In addition, the mean litter proportion of interventricular septal defect was within the range of the WIL Research historical control data for definitive studies, whereas malpositioned atrium has not been previously observed in the WIL Research historical control data. Fetus no. 8086-04 in the control group was also noted with an absent left kidney, ureter, and horn of the uterus and transposition of the great vessels. Another fetus in the same control litter (no. 8086-16) had a malpositioned right testis and epididymis (located more anterior than normal). Fetus no. 8114-01 in the control group also had lobular dysgenesis of the lungs (1 lobe was present bilaterally). No test item-related visceral developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.
Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) were noted for 9, 5, 3, and 3 fetuses in the control, 100, 300, and 1000 mg/kg/day groups, respectively. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test item-related because they occurred infrequently in the test item-treated groups, at a higher frequency in the control group, and in a manner that was not dose-related.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
Although the mean litter proportion of pre-implantation loss was unaffected by test itemadministration at all dosage levels, the mean number of implantation sites in the 1000 mg/kg/day group (13.6 sites/dam) was significantly (p<0.05) lower than the concurrent control group value (15.3 sites/dam). As a result, the mean number of viable fetuses in the 1000 mg/kg/day group (12.9 per litter) was lower than the concurrent control group (14.4 per litter). The lower mean number of implantation sites was attributed a lower number of corpora lutea in the 1000 mg/kg/day group (15.2/dam) compared to the control group (16.5/dam) and the mean value in the WIL Research historical control data (17.0 ± 0.91/dam). Because ovulation occurred prior to initiation of test item administration on gestation day 1, the lower number of implantation sites in the 1000 mg/kg/day group was not considered to be test item-related. The mean litter proportion of postimplantation loss in this group was unaffected by test item
administration. Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group (3.8, 3.6, and 3.7 g, respectively) were slightly lower than the concurrent control group values (4.0, 3.8, and 3.9 g, respectively); the difference for females was significant (p<0.05). However, all high-dosage group values were equal to the respective mean values in the WIL Research historical control data; therefore, the slightly lower mean fetal body weights in the 1000 mg/kg/day group were not considered test item-related. Mean fetal body weights in the 100 and 300 mg/kg/day groups were similar to the control group values; none of the differences were statistically significant. Intrauterine growth and survival was unaffected by test item administration at dosage levels of 100 and 300 mg/kg/day. Parameters evaluated included pre-implantation loss, postimplantation loss, live litter size, and fetal sex ratios.

FETAL MORPHOLOGICAL DATA
The numbers of fetuses (litters) available for morphological evaluation were 359(25), 349(25), 312(23), and 271(21) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Malformations were observed in 5(2), 2(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

EXTERNAL MALFORMATIONS AND VARIATIONS
External malformations were noted in 1 fetus in the 100 mg/kg/day group. Fetus no. 8092-05 in the 100 mg/kg/day group was observed with macroglossia and a palate with a high arch. Based on the occurrence in a single fetus and the absence of a dose-response relationship, these malformations were not considered test item-related. No external developmental variations were observed in fetuses in this study.

VISCERAL MALFORMATIONS AND VARIATIONS
Visceral malformations were noted in 3(2), 0(0), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Interventricular septal defects (≤ 2-mm in diameter opening in the anterior or posterior portion of the septum) were noted for fetus nos. 8086-04 and 8114-01 in the control group and fetus no. 8074-09 in the 1000 mg/kg/day group. Fetus no. 8074-09 in the 1000 mg/kg/day group also had a malpositioned atrium (the left atrium was located more dorsal than normal). The occurrences of interventricular septal defect and malpositioned atrium in the 1000 mg/kg/day group were not attributed to the test item because they occurred in a single fetus and/or similarly in the concurrent control group and the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group. In addition, the mean litter proportion of interventricular septal defect was within the range of the WIL Research historical control data for definitive studies, whereas malpositioned atrium has not been previously observed in the WIL Research historical control data. Fetus no. 8086-04 in the control group was also noted with an absent left kidney, ureter, and horn of the uterus and transposition of the great vessels. Another fetus in the same control litter (no. 8086-16) had a malpositioned right testis and epididymis (located more anterior than normal). Fetus no. 8114-01 in the control group also had lobular dysgenesis of the lungs (1 lobe was present bilaterally). No test item-related visceral developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) were noted for 9, 5, 3, and 3 fetuses in the control, 100, 300, and 1000 mg/kg/day groups, respectively. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test item-related because they occurred infrequently in the test item-treated groups, at a higher frequency in the control group, and in a manner that was not dose-related.

SKELETAL MALFORMATIONS AND VARIATIONS
Skeletal malformations were noted in 2(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fetus no. 8074-09 in the 1000 mg/kg/day group (previously noted with visceral malformations) had malaligned sternebrae (nos. 2 and 3 [moderate] and 4 and 5 [severe]). Fetus nos. 8086-11 and 8086-13 in the control group and no. 8152-05 in the 100 mg/kg/day group had costal cartilage anomalies (fused, bifurcated, and/or malpositioned costal cartilages). The skeletal malformations noted in the 100 and 1000 mg/kg/day groups were not attributed to the test item because they occurred in single fetuses and/or similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data. No test item-related skeletal developmental variations were noted. Findings observed in the test item-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data for definitive studies.

SUMMARY OF EXTERNAL, VISCERAL, AND SKELETAL EXAMINATIONS
The numbers of fetuses (litters) available for morphological evaluation were 359(25), 349(25), 312(23), and 271(21) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Malformations were observed in 5(2), 2(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test item-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the WIL Research historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test item.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on fetuses

Remarks on result:

other: (NOAEL is the highest dose (limit dose) of 1000 mg/kg bw/day.)

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on maternal toxicity in the 1000 mg/kg/day group, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on a lack of developmental toxicity at all dosage levels, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

The developmental toxicity test was performed according to OECD guideline 414 with GLP compliance.The test item was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.12, 0.35, and 1.16 mL/kg, respectively.

Based on maternal toxicity in the 1000 mg/kg/day group, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on a lack of developmental toxicity at all dosage levels, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity when test item was administered orally by gavage to bred Crl:CD(SD) rats.