3,4-dihydroxy-1-[(Z)-octadec-9-enyl]pyrrolidine-2,5-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 24 October 2010 and 17 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Description : Light brown solid
Date received : 25 August 2010,
Storage conditions : at room temperature
Exp. date : 23 August 2012

Test animals

Species:

rabbit

Strain:

other: New Zealand White [Hra:(NZW)SPF] rabbits

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Age at study initiation:
approximately 5 months.

- Weight at study initiation:
2.9 to 4.5 kg (female) on Gestation Day 0.

- Fasting period before study:
Not applicable.

- Housing:
All animals were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH).

- Use of restrainers for preventing ingestion (if dermal):
Not applicable.

- Diet:
The animals were allowed free access to food

- Water:
Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum.

- Acclimation period:
Not applicable.
Time-mated rabbits were received on gestation day 1, 2, or 3 and received treatment once daily during gestation days 7-28.

ENVIRONMENTAL CONDITIONS

- Temperature (°C):
19°C ± 3°C

- Humidity (%):
50% ± 20%

- Air changes (per hr):
At least ten air changes per hour.

- Photoperiod (hrs dark / hrs light):
12 hours continuous light and 12 hours darkness.

IN-LIFE DATES:
31 October - 23 November 2010 (test substance administration period).

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% carboxymethylcellulose

Details on exposure:

Method of administration:
Gavage

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in 0.5% carboxymethylcellulose.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not applicable.

VEHICLE
- Justification for use and choice of vehicle(if other than water): Most suitable

- Concentration in vehicle:
0, 10, 30 and 80 mg/ml (pH values 7.54, 7.04, 6.51 and 6.04)

- Amount of vehicle (if gavage):
5 ml/kg

- Lot/batch no. (if required):
No data

- Purity:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See attached APPENDIX C - Analyses of Dosing Formulations

A high performance liquid chromatography method using ultraviolet absorbance detection at a wavelength of 210 nm for the determination of the test substance concentration in aqueous formulations containing 0.5% (w/v) carboxymethylcellulose in deionized water and test substance ranging in concentration from 5.00 to 175 mg/mL was validated in a previous study (WIL-168187). In the present study, formulations prepared at target test substance concentrations ranging from 5 to 120 mg/mL were analyzed to assess test substance homogeneity and, following 5 days of room temperature storage, resuspension homogeneity and stability. Also, formulations used for dose administration were analyzed to verify test substance concentration acceptability.

The results of the test substance homogeneity assessment in formulations prepared at target concentrations of 5 and 120 mg test substance/mL met the WIL Research SOP acceptance criteria for homogeneity, i.e., the relative standard deviation (RSD) for the mean concentration was ≤10% at a concentration within the acceptable limits (85% to 115% of target). Assessment of test substance resuspension homogeneity and stability following 5 days of room temperature storage in formulations prepared at target concentrations of 5 and 120 mg test substance/mL met the WIL Research SOP acceptance criteria for resuspension homogeneity, i.e., the RSD for the mean concentration was ≤10%, and stability, i.e., the post-storage concentration was not <90% of the pre-storage value.

The analyzed formulations used for dose administration met the WIL Research SOP acceptance criteria for concentration acceptability for suspension formulations, i.e., the analyzed concentration was 85% to 115% of the target concentration. No test substance was detected in the analyzed vehicle administered to the control group.

Details on mating procedure:

- M/F ratio per cage:
Males – Not applicable

Pregnant females were individually housed

- Length of cohabitation:
Not applicable

- Proof of pregnancy:
Time-mated rabbits were received on gestation day 1, 2, or 3; a breeding record was provided by the supplier. Each female was examined by a qualified biologist on the day of receipt. Body weights were recorded on gestation days 4 and 7, and food consumption was recorded during gestation
days 4-7.

- Further matings after two unsuccessful attempts:
Not applicable

- After successful mating each pregnant female was caged:
Not applicable (females were housed individually throughout the study).

- Any other deviations from standard protocol: See – Appendix A - Study Protocol and Deviations

PLANNED DEVIATIONS
Protocol Section 7.5.2 states that analyses to demonstrate the stability of the test substance formulations and, for suspensions, the homogeneity of the test substance formulations would be conducted before the initiation of dosing, if possible, at concentrations spanning the range of concentrations to be used on the study. Specific dose levels for this study were not determined at the time of analyses; therefore, concentrations spanning the range intended for use on the study were 25 mg/kg/day to
600 mg/kg/day.

Reason for Deviation: Specific dose levels for this study were not determined at the
time of analyses.

UNPLANNED DEVIATIONS
Protocol Section 5.5 states that the body weights of animals would be between 2.9 kg to 4.5 kg on gestation day 0. The actual body weight range for animals on gestation day 0 was 2.701 kg to 4.282 kg.

Reason for Deviation: To allow for a sufficient number of animals on study, animals were assigned to study with body weights outside of the protocol-specified range.

Protocol Section 6.4 states that kale supplementation and water bottles would be used for individual animal according to WIL SOPs. However, upon discussion with the Study Director, individual animals were supplemented with kale 7-23 November 2010 at the discretion of management personnel rather than according to SOP T1-276.

Reason for Deviation: To improve the health of the animals.

Duration of treatment / exposure:

Duration of treatment

All pregnant females were treated once daily during gestation (Days 7-28)

Frequency of treatment:

Daily

Duration of test:

Receipt of time-mated rabbits on gestation days 1, 2, or 3; with dose administration of the test substance between Day 7 and 28.

No. of animals per sex per dose:

25 female rabbits per dose group (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

Dosage levels were selected based upon results of a previous rabbit dose range-finding study (Toot, Draft; WIL-168187), in which a dosage level of 600 mg/kg/day exceeded the maximum tolerated dosage as evidenced by marked body weight losses and severely reduced food consumption, resulting in early termination of this group. A dosage level of 300 mg/kg/day was tolerated with transient lower mean body weight gains and decrements in food consumption at the initiation of treatment.

Therefore, dosage levels of 50, 150, and 400 mg/kg/day were selected for a prenatal developmental toxicity study of the test substance administered orally by gavage to pregnant New Zealand White rabbits.

- Rationale for animal assignment (if not random):
Random.

- Other:
Not applicable

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule:

All rabbits 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 from the day of receipt through gestation day 29 (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 individual 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 (by supplier), 4, and 7-29 (daily).

FOOD CONSUMPTION:
Yes
Individual food consumption was recorded on gestation days 4-29.

FOOD EFFICIENCY:
No

WATER CONSUMPTION:
No

POST-MORTEM EXAMINATIONS: Yes
Maternal animals:

A gross necropsy was performed on females that died or were euthanized in extremis during the course of the study. The number and location of
implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally and retained in 10% neutral-buffered formalin. The females and all other products of conception were then discarded.

Females killed at study termination were euthanized on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. 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 comments, 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). 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.

- Organs examined:
Gravid uterine weight was recorded at termination.

Histopathology:
No
Not applicable

OTHER:
No
Not applicable

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:
No

- Number of late resorptions:
Yes

- Other:
The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Viable foetuses – sex, head, eyes, palate, external orifices, viscera, heart, major blood vessels and kidneys.
The heads from all fetuses were examined by a midcoronal slice.
All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S. 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).

GROSS EXAMINATION OF DEAD PUPS:
Late resorptions examinations included crown-rump measurements, degrees of autolysis and gross examinations.

POST-MORTEM EXAMINATION

The detailed external examination of each fetus included, examination of eyes, palate, external orifices. Each viable fetus was also subjected to a examination of the viscera, heart and major blood vessels. The sex of each fetus was determined by internal examination. Kidneys were examined and graded for renal papillae development. Heads from all fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100 ethyl alcohol.

Late resorptions examinations included crown-rump measurements, degrees of autolysis and gross examinations.

Statistics:

The following parameters were subjected to statistical analysis:

See attachment - STATISTICAL ANALYSES

Indices:

See section "Any other information on results including Tables".

Historical control data:

See Attachment - Appendix F - Historical Control Data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
See attached Summary results Tables (S1 to S15) and Appendix G (Individual results Tables A1 to A15)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)


(Clinical signs)
In the 400 mg/kg/day group, test substance-related higher incidences of decreased defecation, soft stool, and small feces (for 21, 19, and 9 females, respectively) were noted compared to the control group beginning on gestation day 8 and continuing until gestation day 29. In addition, an increased incidence of soft stool was noted in the 150 mg/kg/day group relative to the control group; however, since this observation did not correspond to body weight or food consumption effects at this dosage level, it was not considered to be adverse. Decreased defecation was also noted in the 50 and Page 31 of 350 150 mg/kg/day groups primarily beginning on gestation day 22; however, instances were generally sporadic and occurred at a comparable frequency in the control group. The frequencies of these excreta-related findings are presented in the following table.

(Mortality)
Treatment-related mortalities occurred in the 400 mg/kg/day dose group.

Female nos. 62560 and 62569 in this group were euthanized in extremis or found dead, respectively, on gestation day 16. These females lost 10.1% and 11.6% of their gestation day 7 body weights, respectively, and consumed ≤15 g of food per day beginning on gestation day 8 and continuing through death/euthanasia.

In addition, female no. 62564 in this group was euthanized in extremis on gestation day 27. This female lost 8.0% of its gestation day 7 body weight and had reduced food consumption (≤5 g/day) beginning on gestation day 15. Clinical findings noted for these females at the daily examinations were excreta-related and consisted of decreased defecation, soft stool, mucoid feces, and small feces, which were generally observed beginning on gestation day 8 and continuing until death/euthanasia.

There were no other unscheduled deaths.

BODY WEIGHT AND GRAVID UTERINE WEIGHTS (PARENTAL ANIMALS)
See attached Tables S4 to S5 and Individual Data Tables A5 to A7

In the 400 mg/kg/day group, a mean body weight loss was noted during gestation days 7-10 followed by lower mean body weight gains during gestation days 10-13 and 13-20; the differences from the control group were significant (p<0.05 or p<0.01) during gestation days 7-10, 10-13, and 14-15. As a result of these changes, mean body weight in the 400 mg/kg/day group was up to 6% lower, during the treatment period.

However, a higher (not statistically significant) mean body weight gain was noted in this group during gestation days 20-29 when compared to the control group. Consequently, overall mean body weight gain in this group was only slightly lower (not statistically significant) than the control group, and mean body weight was comparable to the control group on gestation day 29. Furthermore, a lower (not statistically significant) mean net body Page 32 of 350 weight gain was observed at 400 mg/kg/day when compared to the control group, but mean net body weight and gravid uterine weight were comparable.

FOOD CONSUMPTION (PARENTAL ANIMALS)
See attached Tables S7 to S8, Individual Data Tables A8 to A9 and Appendix D

In the 400 mg/kg/day group, mean food consumption, evaluated as g/animal/day and g/kg/day, was significantly (p<0.01) lower than the control group during gestation days 7-10, 10-13, and 13-20 and comparable to the control group during gestation days 20-29, which corresponded to mean body weight changes in this group. As a result, mean food consumption was significantly (p<0.01) lower when the overall treatment period (gestation days 7-29) was evaluated and was considered to be test substance-related.

Food consumption at 50 and 150 mg/kg/day was unaffected by test substance administration. The only significant (p<0.05) difference from the control group was lower food consumption noted in the 50 mg/kg/day group during gestation days 21-22 that was transient and not dose-related.

Food consumption at 50 and 150 mg/kg/day was unaffected by test substance administration. The only significant (p<0.05) difference from the control group was lower food consumption noted in the 50 mg/kg/day group during gestation days 21-22 that was transient and not dose-related.

NECROPSY DATA (PARENTAL ANIMALS)
See attached Table S9 and Individual Data Table A10

In the 400 mg/kg/day group, female nos. 62569 and 62560 were found dead and euthanized in extremis, respectively, on gestation day 16; these females were internally normal and had normally developing implantations in utero. In addition, female no. 62564 in this group was euthanized in extremis on gestation day 27; this female had 2 late resorptions and was noted with cystic oviducts, which was not considered test substance-related because it was noted at a comparable frequency in the control group.

All other females survived to the scheduled necropsy on gestation day 29. Macroscopic findings observed in the test substance-treated groups, including cystic oviducts, accessory spleens, dark red areas of the lungs, and pale liver, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related and were not considered to be related to test substance administration. With the exception of 1, 2, and 2 females in the 50, 150, and 400 mg/kg/day groups, respectively, all females were determined to be gravid. Since implantation occurs prior to the initiation of test substance administration on gestation day 6, the nongravid pregnancy status is not considered to be a result of the test substance.

GESTATION DAY 29 LAPAROHYSTERECTOMY
See attached Tables S10 to S11, Individual Data Tables A11 to A13 and Appendix F

Mean fetal weights (male, female, and combined) in the 400 mg/kg/day group were up to 9.9% lower (significant at p<0.05 or p<0.01), relative to the control group. Although Page 34 of 350 mean fetal weights in this group were within the range of the WIL historical control data for definitive studies, they were considered to be test article-related. Mean fetal weights in the 50 and 150 mg/kg/day groups were unaffected by test substance administration.

Intrauterine survival was unaffected by test substance administration at dosage levels of 50, 150, and 400 mg/kg/day. Parameters evaluated included postimplantation loss, live litter size, 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, not statistically significant, and did not occur in a dose-related manner.

FETAL MORPHOLOGICAL DATA
See attached Tables S12 to S15, Individual Data Table A14 and Appendix F

The numbers of fetuses (litters) available for morphological evaluation were 221(25), 190(24), 198(23), and 176(20) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 3(3), 0(0), 1(1), and 0(0) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

EXTERNAL MALFORMATIONS AND VARIATIONS

No external malformations or developmental variations were noted for any fetuses in this study. However, an open eyelid was observed for a late resorption (no. 3) in the litter of female no. 62576 in the 150 mg/kg/day group. Because this finding was not observed in a high-dose fetus, it was not considered related to test substance administration

VISCERAL MALFORMATIONS AND VARIATIONS

Visceral malformations were noted in 1 fetus in the 150 mg/kg/day group and 2 fetuses in the control group. In the 150 mg/kg/day group, fetus no. 62528-04 was noted with a bulbous aorta (ascending and aortic arch). Because this finding was not observed in the Page 35 of 350 high-dose group, it was not considered to be related to test substance administration. In the control group, fetus nos. 62527-08 and 62573-04 were noted with retroesophageal aortic arch (aortic arch coursed retroesophageal immediately following left carotid artery and rejoined in normal position adjacent to ductus arteriosus) and hydrocephaly (increased cavitation of both lateral ventricles and the third ventricle), respectively.

The developmental variation major blood vessel variation was noted in 7(5), 10(7), 7(4), and 19(6) fetuses (litters) in the control, 50, 150, and 400 mg/kg/day groups, respectively.

Although the mean litter proportion of this variation (10.1% per litter) at 400 mg/kg/day was higher than the concurrent control group (3.3% per litter), the difference was not statistically significantly different from the concurrent control group, and the value was within the range of WIL historical control data (0.0% to 13.5% per litter). Furthermore, the number of litters affected at 50, 150, and 400 mg/kg/day (7, 4, and 6, respectively) was comparable to the control group (5 litters) and did not occur in a dose-related manner. Therefore, this finding was not considered to be test substance-related.
Additional visceral developmental variations consisted of accessory spleen(s), absent or small gallbladder, extra papillary muscle of the heart, retrocaval ureter, accessory lobule(s) of the liver, and hemorrhagic ring around the iris. The mean litter proportions of these findings were not statistically significantly different when compared to the concurrent control group, were within the range of the WIL historical control data for definitive studies, and/or occurred in a manner that was not dose-related.

SKELETAL MALFORMATIONS AND VARIATIONS

There were no skeletal malformations observed in any fetus in the 50, 150, or 400 mg/kg/day groups. The only skeletal malformation observed in this study was costal cartilage anomaly (costal cartilage arose from right 7th cervical rib fused to right costal cartilage no. 1 and associated with sternum in normal no. 1 position) in a single control group fetus (no. 62532-08).

Skeletal developmental variations, consisting of 13th full rib(s), sternebra(e) nos. 5 and/or 6 unossified, 7th cervical rib(s), 13th rudimentary rib(s), 27 presacral vertebrae, thyoid arch(es) bent, sternebra(e) malaligned (slight or moderate), sternebrae with thread-like attachment, sternebra(e) nos. 1, 2, 3, and/or 4 unossified, accessory skull bone(s), extra site of ossifiecation ventral to cervical centrum no. 2, extra site of ossification anterior to sternebra no. 1, 25 presacral vertebrae, and 7th sternebra, were noted similarly in the concurrent control group and/or in a manner that was not dose-related. Furthermore, when evaluated on a mean litter proportion basis, incidences were within the range of WIL historical control data for definitive studies and differences from the concurrent control group were not statistically significant. Therefore, these skeletal developmental
variations were not considered to be test substance-related.

SUMMARY OF EXTERNAL, VISCERAL, AND SKELETAL EXAMINATIONS

The numbers of fetuses (litters) available for morphological evaluation were 221(25), 190(24), 198(23), and 176(20) in the control, 50, 150, and 400 mg/kg/day groups, respectively. Malformations were observed in 3(3), 0(0), 1(1), and 0(0) fetuses (litters) in the same respective groups, and were considered to be spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the concurrent control group were noted.

Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency comparable to that in the concurrent control group, did not occur in a dose-related manner, and/or were within the WIL historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Remarks on result:

other:

Remarks:

maternal toxicity was evidenced by moribundity and mortality and excreta-related clinical findings, mean body weight losses, lower mean body weight gains, and reduced food consumption in the 400 mg/kg/day group. In addition, reduced mean fetal weights were observed at 400 mg/kg/day. Therefore, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal development when the test substance was administered orally by gavage to pregnant New Zealand White rabbits

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Not applicable

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

Remarks on result:

other:

Remarks:

Mean fetal weights (male, female, and combined) in the 400 mg/kg/day group were up to 9.9% lower (significant at p<0.05 or p<0.01), relative to the control group. Although mean fetal weights in this group were within the range of the WIL historical control data for definitive studies, they were considered to be test article-related. Mean fetal weights in the 50 and 150 mg/kg/day groups were unaffected by test substance administration.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

See attached Tables and Appendices Litter Indices

1. Group Mean Litter Basis:
Post implantation Loss/Litter =	No. Dead Fetuses,
	Resorptions (Early/Late)/Group
	No. Gravid Females/Group
2. Proportional Litter Basis:
Summation Per Group (%) =	Sum of Post implantation Loss/Litter (%)
	No. Litters/Group
Where:
Post implantation Loss/Litter (%) =	No. Dead Fetuses Resorptions (Early/Late)/Group	x 100
	No. Implantation Sites/Litter

 

Applicant's summary and conclusion

Conclusions:

Summary of Results

In the 400 mg/kg/day group, 1 female was found dead on gestation day 16, and 2 females were euthanized in extremis on gestation days 16 and 27. These deaths were considered to be related to test substance-administration as they occurred following mean body weight losses, lower food consumption, and excreta-related clinical findings. Test substance-related clinical findings for surviving females in the 400 mg/kg/day group consisted of decreased defecation, mucoid feces, and soft stool and were generally observed beginning on gestation day 8 and continuing until euthanasia. No test substance-related clinical observations were noted in the 50 and 150 mg/kg/day groups.

Mean body weight losses and lower mean body weight gains during gestation days 7-20 and lower food consumption throughout the treatment period were noted at 400 mg/kg/day. As a result, mean body weight at 400 mg/kg/day was up to 6% lower than the control group during gestation, and a lower mean net body weight gain was observed at the scheduled necropsy. However, because mean body weight gain in this group was higher than the control group during the latter part of the treatment period (gestation days 20-29), mean body weight was comparable to the control group by gestation day 29. Mean gravid uterine weights at all dosage levels and mean maternal body weights, body weight gains, net body weights, net body weight gains, and food consumption in the 50 and 150 mg/kg/day groups were unaffected by test substance administration.

There were no remarkable internal findings noted at 50, 150, and 400 mg/kg/day. Test substance-related reduced mean fetal weights (up to 9.9%) were noted at 400 mg/kg/day when compared to the control group. Intrauterine growth at 50 and 150 mg/kg/day and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration.

No test substance-related malformations or developmental variations were observed in any fetuses.

In conclusion, maternal toxicity was evidenced by moribundity and mortality and excreta-related clinical findings, mean body weight losses, lower mean body weight gains, and reduced food consumption in the 400 mg/kg/day group. In addition, reduced mean fetal weights were observed at 400 mg/kg/day. Therefore, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal development when the test substance was administered orally by gavage to pregnant New Zealand White rabbits.

Executive summary:

OBJECTIVE

The objective of the study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the period from implantation to one 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 and developmental toxicity.

 

STUDY DESIGN

 

The test substance in the vehicle, 0.5% carboxymethylcellulose (CMC), was administered orally by gavage to 3 groups of 25 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from gestation days 7 through 28. Dosage levels were 50, 150, and 400 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 25 time-mated females received the vehicle (0.5% CMC) on a comparable regimen. The females were approximately 5.5 months 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 29, a laparohysterectomy was performed on each surviving 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.

 

CONCLUSION

 

Maternal toxicity evidenced by moribundity and mortality and excreta-related clinical findings, mean body weight losses, lower mean body weight gains, and reduced food consumption in the 400 mg/kg/day group. In addition, reduced mean fetal weights were observed at 400 mg/kg/day. Therefore, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal development when the test substance was administered orally by gavage to pregnant New Zealand White rabbits.