Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-27 to 2015-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The United States EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (16-0ct-1989 and 18-Sep-1989, respectively), and the OECD Principles of GLP [C(97) 186/Final], 26-Nov-1997.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Substance type: Alkylamine salt of alkyl phosphoric acid
- Physical state: Yellow, translucent viscous liquid
- Analytical purity: 100 %
- Purity test date: February, 24, 2015
- Expiration date of the lot/batch: 24-Feb-2017
- Stability under test conditions: stable
- Storage condition of test material: The test substance was stored at room temperature, and was considered stable under this condition. A reserve sample of the test substance was collected and stored in the WIL Research Archives.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 79 days old upon receipt; 13 weeks old when paired for breeding.
- Weight at study initiation: The day following receipt, all animals were weighed and clinical observations were recorded (without further details). At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. Body weight values ranged from 225 g to 307 g on gestation day 0.
- Fasting period before study: no
- 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 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. 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 (e.g. ad libitum): ad libitum (The basal diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002)
- Water (e.g. ad libitum): ad libitum (Reverse osmosis-purified (on-site) drinking water)
- Acclimation period: a minimum of 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 71°F ± 5°F (22°C ± 3°C); Actual mean daily temperature ranged from 68.0°F to 70.3°F (20.0°C to 21.3°C)
- Humidity: 50% ± 20%; mean daily relative humidity ranged from 53.0% to 63.8
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 June 2015 Experimental starting date (animal receipt) To: 09 July 2015 (laparohysterectomy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
(peanut) oil, NF
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance 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. Dosing formulations were prepared at the test substance concentrations indicated in the table 1.

The test substance 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 substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance is well miscible in peanut oil.
- Concentration in vehicle: 0, 20, 60, 100 and 200 mg/mL (see also table 1)
- Amount of vehicle (if gavage): 5 mL/ kg bw (see also table 2)
- Lot/batch no. (if required): 2EA0347, exp. date: 31-Jan-2016
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity, resuspension homogeneity, and stability of the test substance in the vehicle for 8 days of room temperature storage were previously demonstrated at concentrations ranging from 10 to 200 mg/mL (Sen, 2015, WIL-168239).
Samples for concentration and/or homogeneity determination were collected from the middle stratum of the first and last control group dosing formulations and from the top, middle, and bottom strata of the first and last test substance dosing formulations. One set of samples from each collection was subjected to the appropriate analyses (except as noted above). All remaining samples were stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection (Sen, 2015, WIL-168239).
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused: 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.
- M/F ratio per cage: 1/1
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
during gestation days 6-19
Frequency of treatment:
once daily
Duration of test:
from 02-Jun-2015 (experimental starting date (animal receipt) until 20-Aug-2015 (experimental termination/completion date (last fetal skeletal examination)
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 500 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous OECD 421 study in Sprague Dawley rats (Thorsrud, 2013). Treatment-related effects were observed at dosages of 500 and 1000 mg/kg/day following a duration of exposure that was more than twice as long as the duration in the current study. Body weight effects were limited to females. Occurrences of mean body weight decreases were sporadic during the early weeks of the study and did not become consistent and pronounced until the end of the gestation period. No animals were found dead during the course of the study, although 2 females in the 1000 mg/kg/day group were euthanized in extremis at the time of parturition on gestation days 21 or 22. The NOAEL was 150 mg/kg/day. As a result, a high-dosage level of 1000 mg/kg/day and lower dosage levels were chosen to evaluate the dose-response of the test substance in the current study.
- Rationale for animal assignment: randomised. 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.
- Other: Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
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.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: 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).
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:
- External examinations: Yes: [all per litter] 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.
- Soft tissue examinations: Yes: [all per litter] 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. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter] 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. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-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.
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 substance-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. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Indices:
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group;

2. 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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at 1000 mg/kg bw

Details on maternal toxic effects:
- All females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20;
- Clinical signs:
- 1000 mg/kg bw: red material around the nose and mouth, decreased defecation, pale feces, and yellow material around the urogenital (adverse effects); 3 females: hunched posture on gestation day 20; 1 female: clonic convulsions on gestation day 19 (transient effects, not attributed to test substance administration);
- 100, 300, and 500 mg/kg/day groups: clear material around the mouth and red material around the nose during the gestation days 7-13 (test substance-related, non-adverse effects);
- Other findings: hair loss on the forelimbs (all treatment groups, the control group; no dose-related effect).

Body weights and gravid uterine weights:
- 1000 mg/kg bw: test substance-related mean body weight losses (4.6% to 19.4%) and lower mean body weight gains with corresponding lower mean food consumption; lower mean body weights, net body weight, and net body weight gains;
- 500 mg/kg/day group: a lower mean body weight gain with corresponding lower mean food consumption during gestation days 6-20 (non-adverse effect);
- 100 and 300 mg/kg/day groups: no effects on mean body weights and body weight gains;
- 100, 300, and 500 mg/kg/day: no effects on mean net body weights, net body weight gains, food consumption, or gravid uterine weights;

Necropsy findings:
- All treatment groups: There were no test substance-related macroscopic findings noted at any dosage level.

Intrauterine growth and survival:
- 1000 mg/kg bw: mean male, female, and combined fetal weights were 15.8%, 13.9%, and 16.2% lower, respectively, than the concurrent control group (the differences were significant: p< 0.01); gravid uterine weights are significantly lower than in the control group (p< 0.01); pre- and postimplantation loss, live litter size, and fetal sex ratios (unaffected);
- 100, 300, and 500 mg/kg/day: no effects.
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)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at 1000 mg/kg bw

Details on embryotoxic / teratogenic effects:
- 1000 mg/kg bw: higher mean total proportion of skeletal developmental variations (unossification of sternebra(e) no. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4 and reduced ossification of the vertebral arches; test substance-related effects, secondary effects to the reduced fetal body weights noted at this dosage level);
- 100, 300, and 500 mg/kg/day: no test substance-related fetal malformations and variations were observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 3. Results of Homogeneity and Concentration Analyses

 

Group 2
(20 mg/mL)

Group 3
(60 mg/mL)

Group 4
(100 mg/mL)

Group 5
(200 mg/mL)

Homogeneity and Concentration Assessment of the 19-Jun_2015 Formulations (Back-Up Samples)

Mean Concentration (mg/mL)

19.5

59.9

96.4

202

RSD (%)

1.3

3.6

3.1

2.9

Mean % of Target

97.4

99.8

96.4

101

Homogeneity and Concentration Assessment of the 02-Jul-2015 Formulations

Mean Concentration (mg/mL)

22.6

69.0

109

220

RSD (%)

2.3

2.3

2.1

3.2

Mean % of Target

113

115

109

110

Fetal morphological data

External malformations and variations

External malformations were noted for 4(1) and 1(1) fetuses (litters) in the 100 and 300 mg/kg/day groups, respectively. Fetuses in the 100 mg/kg/day group had vertebral agenesis which consisted of a short tail. Skeletally, these malformations consisted of fewer than normal vertebrae in the cervical, thoracic, and/or lumbar regions; fewer than normal ribs; misshapen, malpositioned, fused, unossified and/or absent vertebrae; small, misshapen, and malpositioned ribs; costal cartilages may or may not associate with the sternum; and only 1 sternebra present. In the 300 mg/kg/day group, one fetus had a cleft palate and mandibular micrognathia. Skeletally, the cleft palate was confirmed as the palatine plates not joined along the entire length and mandibular micrognathia consisted of a mandible smaller than normal and fused along the mandibular symphysis. Because these external malformations were observed in a single fetus or litter and did not occur in a dose-related manner, they were not considered test substance-related.

There were no external developmental variations noted for fetuses at any dosage level.

Visceral Malformations and Variations

Hydrocephaly was noted for one fetus in the 100 mg/kg/day group. This visceral malformation consisted of increased cavitation of the lateral, bilateral, and third ventricles. In the absence of a dose-response, this visceral malformation was not attributed to maternal test substance administration. Visceral developmental variations observed in the test substance-treated groups included renal papilla(e) not developed and/or distended ureter(s), accessory lobule(s) in the liver, small and pale spleen, hemorrhagic ring around the iris, and short brachiocephalic trunk. These visceral developmental variations were noted in single fetuses, similarly in the concurrent control group, in a manner that was not dose-related, and/or the mean litter proportions were within the WIL Research historical control data. No relationship to the test substance was evident for any visceral developmental variation. Renal papilla(e) not fully developed (Woo and Hoar Grade 1) were noted for 2, 9, 4, and 7 fetuses in the control, 100, 300, and 500 mg/kg/day groups, respectively. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it 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), 4(3), 1(1), 0(0), and 0(0) fetuses (litters) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively. One fetus in the 300 mg/kg/day group had 6 cervical vertebrae (25 presacral vertebrae). In the 100 mg/kg/day group, one fetus had a costal cartilage anomaly (costal cartilage no. 1 bifurcated, fused, and associated with the sternum in normal position no. 1) and another had a rib anomaly (fused ribs). In addition, two fetus had skull anomalies that consisted of absent exoccipital bones; these fetuses were also noted with an external malformation of vertebral agenesis. These skeletal malformations were noted for single fetuses or litters, were not observed in a dose-related manner, the mean litter proportions were not statistically significantly different from the concurrent control group, and the mean litter proportions of these findings were within the WIL Research historical control data ranges. Therefore, the skeletal malformations observed in the 100 and 300 mg/kg/day groups were not considered to be test substance-related.

Test substance-related skeletal developmental variations noted in the 1000 mg/kg/day group included increased mean litter proportions of reduced ossification of the vertebral arches, unossification of sternebra(e) no. 5 and/or 6, and unossification of sternebra(e) nos. 1, 2, 3, and/or 4 (see Table 4). The differences were generally significant (p< 0.01) when compared to the concurrent control group and the mean litter proportions exceeded the maximum mean values in the WIL Research historical control data. Furthermore, these skeletal developmental variations were indicative of developmental delay and considered secondary to the reduced fetal body weights noted at 1000 mg/kg/day.

Other skeletal developmental variations observed in the test substance-treated groups consisted of cervical centrum no. 1 ossified, 14th rudimentary rib(s), 14th full rib(s), 7th cervical rib(s), reduced ossification of the 13th rib(s) and skull, unossification of the pubis, vertebral centra, and hyoid, sternebra(e) slightly or moderately malaligned, 27 presacral vertebrae, bent rib(s), and 25 presacral vertebrae. These findings occurred infrequently or at a frequency similar to the concurrent control group, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL Research historical control data range, and therefore these developmental variations were not considered to be test substance-related.

Table 4. Test Substance-Related Skeletal Developmental Variations

Dosage (mg/kg/day)

Absolute no. (% per litter)

WIL HC Mean (Range; % per litter)

0a

100

300

500

1000

Skeletal Developmental Variations

Reduced ossification of the vertebral arches

0 (0.0)

1 (0.3)

3 (1.0)

2 (0.6)

77 (20.3)++

0.2 (0.0 - 2.0)

Sternebra(e) nos. 5 and/or 6 unossified

44 (12.1)

71 (19.4)

53 (17.1)

54 (15.6)

166 (46.3)++

6.3 (0.0 - 26.1)

Sternebra(e) nos. 1, 2, 3, and/or 4 unossified

3 (0.9)

2 (0.7)

4 (1.4)

1 (0.3)

8 (2.1)

0.2 (0.0 -1.5)

a= Vehicle control.

WIL HC = WIL Research developmental historical control data.

++= Statistically significant at 0.01 compared to the control group using Dunn’s test.

Summary of External, Visceral and Skeletal Malformations

A significantly (p<0.05) higher total mean litter proportion of fetal developmental variations was observed in the 1000 mg/kg/day group (64.0% per litter) compared to the concurrent control group (40.9% per litter). This was due to the significantly (p<0.05) higher percent per litter of skeletal developmental variations observed in the 1000 mg/kg/day group (63.7% per litter). Test substance-related increased incidences of skeletal developmental variations in the 1000 mg/kg/day group consisted of reduced ossification or unossified bones (unossified sternebra(e) no. 5 and/or 6, unossified sternebra(e) nos. 1, 2, 3, and/or 4, and reduced ossification of the vertebral arches) which was indicative of a developmental delay and considered secondary to the lower fetal body weights observed at this dosage level.

No test substance-related fetal malformations were observed at any dosage level and no test substance-related developmental variations were noted in the 100, 300, or 500 mg/kg/day groups.

Applicant's summary and conclusion

Conclusions:
Based on adverse clinical findings and mean body weight losses and lower mean body weight gains with corresponding decreased food consumption observed at 1000 mg/kg/day, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on decreased mean fetal body weights with an associated increase in the mean litter proportions of skeletal developmental variations related to reduced ossification at 1000 mg/kg/day, a dosage level of 500 mg/kg/day was considered to be the NOAEL for embryo/fetal development when the test material was administered orally by gavage to bred Crl:CD(SD) rats. Test substance-related increased mean litter proportions of skeletal developmental variations consisted of reduced ossification or unossified bones which is indicative of developmental delay and was considered secondary to the reduced fetal body weights noted at this dosage level. The embryo/fetal effects were associated with maternal toxicity and the corresponding decrease in food consumption in the dams.
Executive summary:

The study was conducted to determine the potential of the test substance (reaction products of alcohols, C14-18, C18 unsat., esterified with phosphorus pentoxide and salted with amines, C12-14,-tert-alkyl) to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

The test substance, in the vehicle (arachis [peanut] oil) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 100, 300, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 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. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related incidences of red material around the nose and mouth, decreased defecation, pale feces, and yellow material around the urogenital area were noted for the 1000 mg/kg/day group at the daily examinations and/or approximately 1 hour following dose administration generally throughout the treatment period. Test substance-related, non-adverse increased incidences of clear material around the mouth was noted in all test substance-treated groups and red material around the nose was noted for the 100, 300, and 500 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration primarily during the first week of treatment (gestation days 7-13). No other test substance-related clinical findings were noted at the daily examinations or 1 hour following dose administration at any dosage level. However, at the daily examinations 3 females in the 1000 mg/kg/day group had hunched posture on gestation day 20 and 1 female in this group had clonic convulsions on gestation day 19. These observations were considered transient and not attributed to test substance administration.

Test substance-related mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption were noted in the 1000 mg/kg/day group compared to the control group throughout the treatment period. In addition, lower mean body weights, net body weight, and net body weight gains were observed at this dosage level.

In the 500 mg/kg/day group, a lower mean body weight gain with corresponding lower mean food consumption was noted compared to the control group during gestation days 6-9. Mean body weight gains and food consumption in this group were generally similar to the control group throughout the remainder of the treatment period. As a result of the initial body weight decrements, a lower mean body weight gain was noted for the 500 mg/kg/day group when compared to the control group for the overall treatment period (gestation days 6-20). However, the lower mean body weight gains noted for the 500 mg/kg/day group were not of sufficient magnitude to affect mean body weights in this group and therefore were not considered to be adverse. There were no test substance-related effects on mean body weights and body weight gains at 100 and 300 mg/kg/day or mean net body weights, net body weight gains, food consumption, or gravid uterine weights at 100, 300, and 500 mg/kg/day.

There were no test substance-related macroscopic findings noted at any dosage level.

Mean fetal body weights in the 1000 mg/kg/day group were up to 16.2% lower than the control group and corresponded to the lower mean gravid uterine weight observed in this group. Fetal survival in this group was not affected by test substance administration. Intrauterine growth and survival in the 100, 300, and 500 mg/kg/day groups were unaffected by maternal test substance administration. Test substance-related higher mean total proportion of developmental variations were noted in the 1000 mg/kg/day group when compared to the control group due to a test substance-related higher percent per litter of skeletal developmental variations observed in this group. Higher mean litter proportions of unossification of sternebra(e) no. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4 and reduced ossification of the vertebral arches were noted in the 1000 mg/kg/day group compared to the control group. The aforementioned skeletal developmental variations were considered test substance-related and secondary to the reduced fetal body weights noted at this dosage level. No test substance-related fetal malformations were observed at any dosage level and no test substance-related fetal developmental variations were noted at 100, 300, and 500 mg/kg/day.