diethyl 1,4-cyclohexanedicarboxylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January to 09 April 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 22 January 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl cyclohexane-1,4-dicarboxylate

EC Number:

202-347-5

EC Name:

Dimethyl cyclohexane-1,4-dicarboxylate

Cas Number:

94-60-0

IUPAC Name:

dimethyl cyclohexane-1,4-dicarboxylate

Test material form:

liquid

Details on test material:

- Physical state: Clear, colorless liquid
- Storage condition of test material: Stored at room temperature

Test animals

Species:

rat

Strain:

other: Sprague Dawley [Crl:CD(SD)]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at receipt: Approximately 80 days
- Weight at study initiation: 214-276 g on gestation day 0
- Housing: 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). Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material.
- Diet: Basal diet (PMI Nutrition International, LLC Certified Rodent LabDiet® 5002), ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.0-70.8 °F (21.1-21.6 °C)
- Humidity: 39.3-50.5 %
- Air changes: A minimum of 10 fresh air changes per hour
- Photoperiod: Fluorescent lighting provided illumination for a 12 h light / 12 h dark photoperiod

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.

VEHICLE
- Concentration in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to demonstrate the stability, homogeneity, and resuspension homogeneity of the test item formulations following 11 days of room temperature and refrigerated storage at target concentrations of 10 and 250 mg/mL were assessed in a previous study (Stinson, 2015, WIL-387057).
- Samples for homogeneity determinations were collected from the top, middle, and bottom strata of the first 50 and 200 mg/mL dosing formulations prepared at the smallest and largest formulation volumes prepared during the study. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from these same dosing formulations following refrigerated and room temperature storage for 11 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the first and last dosing formulations at each dosage level (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses.
- All remaining samples were stored refrigerated (approximately 2-8 °C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection (Stinson, 2015, WIL-387057).

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Selected females were approximately 13 weeks when paired for breeding
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage referred to as Day 0 of pregnancy.

Duration of treatment / exposure:

From gestation days 2 through 19

Frequency of treatment:

Once daily

Duration of test:

From gestation days 2 through 19.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 bred females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were determined from results of a previous OECD 421 dietary study in rats. Dietary exposure to levels of DMCD of 0, 0.15, 0.45, or 1.5 % resulted in dosage levels of 0, 111, 351, or 1124 mg/kg bw/day for female rats over the course of the study. Reductions in feed consumption, body weights, and/or rate of body weight gain were noted in the female high-dosage group during the first 7 days of dietary consumption and feed consumption was decreased in the mid- and high-dosage groups on gestation day 7. Mean body weight and body weight gain was reduced in the high-dosage group on gestation day 20. No other effects were noted that were pertinent in setting dosage levels for the current OECD 414 study. It is not known if the reductions in the highest dosage group during the first few days of dosing were due to palatability issues with the treated feed. Based on these results, dosage levels were selected to test the limit dose and below as required by the testing guideline of this study.
- Rationale for animal assignment: 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 stock colony or euthanized by carbon dioxide inhalation and discarded.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 2-20 (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 2-6, 6-9, 9-12, 12-15, 15-20, and 2-20.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on gestation days 0 and 2-20 (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 a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20; All females were euthanized on gestation day 20 by carbon dioxide inhalation.
- The cranial, 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.
- Organs examined: Following organ weight collection, the brain, liver, spleen, and thymus gland were preserved in 10 % neutral-buffered formalin for possible future histopathologic examination; other maternal tissues were preserved 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.

OTHER:
- 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.
- Organ weights: The following organs were weighed from all maternal animals at the scheduled necropsy: brain, liver, spleen, and thymus gland were weighed. Absolute weights and organ to brain weight ratios were reported.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: 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).

Fetal examinations:

- Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region.
- External examinations: Yes; 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.
- Soft tissue examinations: Yes; 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).
- Skeletal examinations: Yes
- Head examinations: Yes: 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). Fetuses were then examined for skeletal malformations and developmental variations.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Group Mean Litter Basis:
Postimplantation Loss/Litter = [No. Dead Fetuses, Resorptions (Early/Late)/Group] / [No. Gravid Females/Group]

Proportional Litter Basis:
Summation Per Group (%) = [Sum of Postimplantation Loss/Litter (%)] / [No. Litters/Group]

Where:
Postimplantation Loss/Litter (%) = ([No. Dead Fetuses, Resorptions (Early/Late)/Litter] / [No. Implantation Sites/Litter]) x 100

Summation per Group (%) = [Sum of Viable Fetuses Affected/Litter (%)] / [No. Litters/Group]
Where:
Viable Fetuses Affected/Litter (%) = [No. Viable Fetuses Affected/Litter] / [No. Viable Fetuses/Litter] x 100

Historical control data:

Results were compared with historical control data of the testing laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
- All females in the control, 250, 500, and 1000 mg/kg bw/day groups survived to the scheduled necropsy on gestation day 20. Test item-related increased incidences of clear and red material around the mouth were noted for all test item-treated groups approximately 1 hour following dose administration generally throughout the treatment period (gestation days 2-20). These clinical findings did not persist to the daily examinations and were therefore not considered adverse. No test item-related clinical findings were noted at the daily examinations at any dosage level. Findings noted in the test item-treated groups, including hair loss on the forelimbs and red material around the nose, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Slightly lower mean body weight gains were noted for all test item-treated groups compared to the control group during gestation days 2 through 6; the differences in the 250 and 1000 mg/kg bw/day groups were significant (p<0.05 or p<0.01) for the gestation day 2-6 interval, but did not occur in a dose-responsive manner. Mean body weight gains in the test item-treated groups were similar to the control throughout the remainder of the treatment period (gestation days 6-20) and when the overall treatment period (gestation days 2-20) was evaluated. In addition, mean body weights in the test item-treated groups were similar to the control group throughout the study. Therefore, the initial lower mean body weight gains observed in the 250, 500, and 1000 mg/kg bw/day groups were considered test item-related; however, because they were transient in nature they were not considered adverse. A significantly (p<0.01) lower mean net body weight gain was noted for the 1000 mg/kg bw/day group. Because there were no effects on mean body weights or mean net body weight in this group, the lower mean net body weight gain in the 1000 mg/kg bw/day group was not attributed to the test item. Mean net body weights and gravid uterine weights in the 250, 500, and 1000 mg/kg bw/day groups and net body weight gains in the 250 and 500 mg/kg bw/day groups were similar to the control group.

MATERNAL FOOD CONSUMPTION
- Maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 250, 500, and 1000 mg/kg bw/day groups was unaffected by test item administration. The only significant (p<0.05 or p<0.01) differences from the control group were lower mean food consumption in the 500 and 1000 mg/kg bw/day groups during gestation day 7-8; these differences were transient and not considered to be test item-related.

MATERNAL NECROPSY DATA
- At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 250, 500, and 1000 mg/kg bw/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. Five, 5, 5, and 3 females in the control, 250, 500, and 1000 mg/kg bw/day groups, respectively, were determined to be nongravid; 3, 2, 2, and 3 of these females, respectively, mated on the first day of pairing. Progressively fewer nongravid females were noted with each subsequent day of mating. The higher incidence of nongravid females was not attributed to the test item as mating occurred prior to test item administration; in addition, it occurred with equal representation in the control group.

ORGAN WEIGHTS
- Slightly lower (not statistically significant) mean absolute thymus gland weights were noted in all test item-treated groups. Due to the lack of statistical significance when compared to the control group and the absence of an effect on the mean thymus gland weight relative to brain weight; the differences in mean absolute thymus gland weights were not considered to be test item-related. Mean absolute and/or relative (to brain weight) brain, liver, and spleen weights in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group.

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
- Intrauterine growth and survival were unaffected by test item administration at dosage levels of 250, 500, and 1000 mg/kg bw/day. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effect at the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL MORPHOLOGICAL DATA
- The numbers of fetuses (litters) available for morphological evaluation were 247(20), 255(20), 270(20), and 283(22) in the control, 250, 500, and 1000 mg/kg bw/day groups, respectively. Malformations were observed in 0(0), 4(2), 2(2), and 0(0) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

EXTERNAL MALFORMATIONS AND VARIATIONS
- Fetus no. 2343-09 in the 250 mg/kg bw/day group that had a vertebral agenesis (with a filamentous tail). Skeletally, all vertebrae posterior to sacral vertebra no. 1 were absent in this fetus. This finding was limited to a single fetus, not observed in a dose-related manner, and the mean litter proportion of the finding was within the range of values in the WIL Research historical control data and was not statistically significantly different from the concurrent control group. Therefore, vertebral agenesis in the 250 mg/kg bw/day group was not attributed to maternal test item administration.
- No other external malformations were noted for fetuses on this study. No external developmental variations were noted for any fetuses in this study.

VISCERAL MALFORMATIONS AND VARIATIONS
- No visceral malformations were noted for fetuses in this study. Visceral developmental variations noted in all group, including the control group, consisted of distended ureters and accessory lobule(s) in the liver. Other findings observed in the test item-treated groups, were noted infrequently and in a manner that was not dose-related. In addition, the mean litter proportions of these findings were not statistically significantly different from the control group and were within the ranges of the WIL Research historical control data. Therefore, the visceral developmental variations noted in 250, 500, and 1000 mg/kg bw/day groups were not considered test item-related.
- Dark red areas on the adrenal glands were noted for fetus nos. 2307-01 and 2307-11 in the control group and fetus no. 2371-03 in the 250 mg/kg bw/day group. In addition, dark red discoloration of the thyroid gland was noted for fetus no. 2307-01 in the control group and renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for fetus nos. 2389-03 and 2389-08 in the 250 mg/kg bw/day group. 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, at similar frequencies in the control group, and/or in a manner that was not dose-related.

SKELETAL MALFORMATIONS AND VARIATIONS
- Skeletal malformations were noted for 0(0), 3(1), 2(2) and 0(0) fetuses (litters) in the control, 250, 500, and 1000 mg/kg bw/day groups, respectively. Rib anomalies were noted for fetus nos. 2389-05, 2389-06, and 2389-08 in the 250 mg/kg bw/day group. This finding consisted of fused ribs. In addition, fetus nos. 2389-05 and 2363-04 in the 250 and 500 mg/kg bw/day groups, respectively, had costal cartilage anomalies that consisted of misshapen or malpositioned costal cartilage. Fetus no. 2378-05 in the 500 mg/kg bw/day group had severely malaligned sternebrae. Skeletal malformations observed in the test item-treated groups occurred in single fetuses or litters, were not observed in a dose-related manner, and/or the mean litter proportions of the findings were within the WIL Research historical control value ranges and not statistically significantly different from the concurrent control group. Therefore, these findings were not attributed to maternal test item administration. No other skeletal malformations were noted at any dosage level.
- Skeletal developmental variations observed all groups, including the control group, consisted of 14th rudimentary rib(s), ossified cervical centrum no. 1, unossified sternebra(e) nos. 5 and/or 6, 7th cervical rib(s), reduced ossification of the 13th rib(s), unossified sternebra(e) nos. 1, 2, 3, and/or 4, and slightly or moderately malaligned sternebra(e). These findings were noted similarly in the control group and/or in a manner that was not dose-related. In addition, the mean litter proportions of these findings were not statistically significantly different from the concurrent control group and were within the WIL Research historical control data ranges. Other skeletal developmental variations were noted in single fetuses or litters, did not occur in a dose-related manner, and/or were within the WIL Research historical control data ranges. No relationship to the test item was evident for any skeletal developmental variation.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect at the highest dose tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85 to 115 %) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).

 

Table 7.8.2/1: Results of Homogeneity Analyses

Homogeneity Assessment	Low Group (50 mg/mL)	High Group (200 mg/mL)
Homogeneity Assessment of the 12-Feb-2015 Formulations
Mean Concentration (mg/mL)	50.5	196
RSD (%)	0.61	0.57
Mean % of Target	101	98.2
11-Day Refrigerated Storage Resuspension Homogeneity Assessment of the 12-Feb-2015 Formulations
Mean Concentration (mg/mL)	51.3	201
RSD (%)	1.1	1.0
Mean % of Target	103	100
11-Day Room Temperature Storage Resuspension Homogeneity Assessment of the 12-Feb-2015 Formulations
Mean Concentration (mg/mL)	51.0	199
RSD (%)	0.37	0.56
Mean % of Target	102	99.7

 

Table 7.8.2/2: Results of Concentration Analyses

Date of Preparation	Mean Concentration, mg/mL (% of Target)
	Group 2 (50 mg/mL)	Group 3 (100 mg/mL)	Group 4 (200 mg/mL)
12-Feb-2015	50.6 (101)	99.9 (99.9)	197 (98.6)
27-Feb-2015	51.3 (103)	104 (104)	197 (98.6)

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL for maternal toxicity and embryo/fetal development is 1000 mg/kg bw/day in rats.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, test substance was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (25/dose) at the dose levels of 0 (corn oil),250, 500, and 1000mg/kg bw/day from gestation days 2 through 19. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. Selected organs were weighed. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

All females in the control, 250, 500, and 1000 mg/kg bw/day groups survived to the scheduled necropsy on gestation day 20. Test item-related clinical findings were limited to dose-dependent clear and red material around the mouth in all test item-treated groups approximately 1 hour following dose administration generally throughout the treatment period. These findings did not persist to the daily examinations (prior to dose administration) on the following day and were not considered to be adverse.

 

Test item-related slightly lower mean body weight gains were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation days 2-6. Mean body weight gains in the test item-treated groups were similar to the control group throughout the remainder of the treatment period (gestation days 6-19) and there were no corresponding effects on mean food consumption or mean absolute body weights. Therefore, the initial body weight decrements in the 250, 500, and 1000 mg/kg bw/day groups were not considered to be adverse. Mean maternal body weights, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 250, 500, and 1000 mg/kg bw/day groups were unaffected by test item administration. No test item-related macroscopic findings or effects on brain, liver, thymus, and spleen weights were noted at any dosage level. Intrauterine growth and survival and fetal morphology in the 250, 500, and 1000 mg/kg bw/day groups were unaffected by maternal test item administration.

 

Based on the lack of adverse maternal or developmental toxicity observed at any dosage level, the NOAEL for maternal toxicity and embryo/fetal development is 1000 mg/kg bw/day in rats.