Chlorothalonil

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Dec 1987 to 22 Jan 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorothalonil

EC Number:

217-588-1

EC Name:

Chlorothalonil

Cas Number:

1897-45-6

Molecular formula:

C8Cl4N2

IUPAC Name:

tetrachlorobenzene-1,3-dicarbonitrile

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

[Hra: (NZW) SPF]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 to 5 months (females), 7 to 9 months (males)
- Weight at study initiation: Females: 3869 g (mean), 3010 to 4779 g (range)
- Fasting period before study: Feed was withheld on the day of arrival.
- Housing: Animals were housed individually, except during mating, in stainless steel suspended cages with wire mesh floors and subcage floor pans in a separate room from other animals in the facility. A stainless steel feeder was affixed to the front of each cage. Feeders were cleaned at weekly intervals.
- Diet: Ad libitum, Certified High-Fiber Rabbit Chow
- Water: Ad libitum, tap water
- Acclimation period: 61 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 to 77 (measured during test; 18 - 25°C)
- Humidity (%): 29 to 69 (measured during test)
- Air changes (per hr): 12.6 (measured during test)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 Dec 1987 To: 22 Jan 1988

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% (w/v) methylcellulose in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose volumes were derived from Day 7 body weights and were adjusted during the remaining treatment period to the most recent body weights. Suspensions for each dose level were prepared daily and stirred prior to and during the dosing procedure.

VEHICLE
- Concentration in vehicle: 2.5, 5.0 and 10.0 mg/mL
- Amount of vehicle: 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to initiation of the study, one pre-test mix was prepared and analyzed for the 2.5 mg/mL and 10.0 mg/mL concentration levels (i.e., Groups II and IV , respectively). As during the dosing procedure, the suspensions were stirred continuously. Seven samples of each of the suspensions were collected by drawing up 5 mL into syringes and dispensing into tared 250 mL amber glass jars. The jars were then weighed to determine the net weight of each sample and the net weight of each sample was added to the label information. Five of the samples were frozen immediately after collection. The other two samples were maintained at room temperature for 24 hours and then frozen. A 100 mL sample of the vehicle was also collected and frozen immediately. Samples were shipped on dry ice for analysis. A duplicate set of seven samples of each suspension (Groups II and IV) and the vehicle were collected in the same manner and maintained frozen at the test facility.

Details on mating procedure:

Natural mating was used. Each female selected for mating was placed into the male 's cage. When coitus was observed, the female was removed and returned to her original cage. Following an interval of one to two hours , the female was placed into the cage of a different male. When coitus was observed with the second male, the female was considered mated and returned to her cage. The day on which coitus was observed with both males was designated Day 0 of gestation.

Duration of treatment / exposure:

From day 7 to day 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Until day 30 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2: Low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3: Mid dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4: High dose

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations checked included: Appearance, behaviour, signs of toxicity, moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Days 0, 7, 10, 13, 16, 19, 24 and 30 of gestation. On Days 7, 10, 13, 16 and 19 of gestation, the detailed physical exams were performed after all animals had been dosed for that day.

BODY WEIGHT:
- Time schedule for examinations: Body weights were recorded three times during the acclimation period. Body weights were also recorded on Days 0, 3, 7, 10, 13, 16, 19, 24 and 30 of gestation. Day 30 body weights are presented as actual and corrected (the actual Day 30 body weight minus the weight of the gravid uterus ).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day
- Time schedule: Food consumption was measured for Days 1, 3, 7, 10, 13, 16, 19, 24 and 29 of gestation.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 30
- Gross postmortem examinations were performed on all mated rabbits including those dying spontaneously during the study. Only abnormal tissues were saved (10% neutral buffered formalin). Females showing signs of premature delivery (presence of fetuses/ pups at Day 26 of
gestation or later ) were killed via intravenous injection of sodium pentobarbital in the marginal ear vein on the day such evidence was observed. Reproductive systems were examined.

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

Data were analyzed between control (Group I) and treated Groups II- IV.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse effect of treatment was evident from the physical examination data.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality rates did not differ statistically between the control or treated groups. No mortality occurred in the control or low-dose group. In the control group, two females delivered prematurely and the remaining 18 females survived to scheduled sacrifice on Day 30 of gestation. In the low-dose group , one female was killed following premature delivery and the remaining 19 females survived to Day 30 gestation scheduled sacrifice. In the mid-dose group, one female died (mortality rate 3 5.0%), two females delivered prematurely and were sacrificed and the remaining 17 females survived to scheduled sacrifice ( Day 30 of gestation ). One female died on Day 10 of gestation after receiving three days of treatment. This female was pregnant as evidenced by 10 uterine implantation sites. At gross postmortem evaluation, this animal had fluid in the lungs and frothy fluid in the trachea, both findings are suggestive of a pulmonary oedema which may or may not have resulted from an intubation error. In the high-dose group, one female died (mortality rate 3 5.0% ), one female delivered prematurely and the remaining 18 females survived to scheduled sacrifice. One female died on Day 13 of gestation after receiving six days of treatment. This female was pregnant as evidenced by 13 uterine implantation sites, determined on the basis of postmortem findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights for each recorded interval during gestation were comparable between the control and each treated group. Mean body weight gains noted for the control and each treated group during the pre- and post-treatment intervals (Days 0-7 and 19-30 of gestation, respectively) were considered comparable. Over the entire treatment period (i.e. , Days 7-19), a comparable mean weight gain was seen in the control, low- and mid-dose groups. The high-dose group experienced a slight mean weight loss during the Day 7-19 treatment period and this difference from control was statistically significant. Likewise, the incidence of females losing weight over the Day 7-19 gestation interval was increased in the high-dose group; these incidences for the control, low-, mid- and high-dose groups were 10% (2/ 20), 10% (2/ 20), 20% (4/ 20) and 47.4% (9/19), respectively
A mean weight loss was noted for the control and each treated group during the Day 7-30 gestation interval using the Day 30 corrected body weights. These data were comparable between the control and each treated group. Thus, no adverse effect of treatment at the 5 or 10 mg/kg bw/day dose levels was evident from body weight data during gestation. At the 20 mg/kg bw/day dose level, a mean weight loss was experienced over the Day 7-19 treatment period and this difference from control data was statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for the pre-treatment (Days 1 and 3), treatment (Days 7, 10, 13, 16 and 19) and post-treatment (Days 24 and 29) intervals were considered comparable between the control, low- and mid-dose groups. In the high-dose group, mean food consumption was comparable to control during the pre- and post-treatment intervals. During the treatment period, mean food consumption in the high-dose group was lower than control for each recording interval; however, only on Day 7 was this difference from control statistically significant. Differences in mean food consumption data for the high-dose group in comparison to control during the treatment period ranged from -8.7% at Day 10 to -35.0% at Day 17. Thus, no adverse effect of treatment on food consumption was evident at the 5 or 10 mg/kg bw/day dose levels. At the 20 mg/kg bw/day dose level, food consumption was reduced from control levels for each recording interval during the treatment period; however, only on Day 7 was this difference from control statistically significant.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For all groups, numerous animals had discolored lungs (primarily red/ tan /brown foci /areas); these tended to be seen more frequently in Groups III and IV as compared to Group I. The incidences of females with discolored lungs for the control, low-, mid- and high-dose groups were 60% (12/ 20), 60% (12/ 20), 70% (14/ 20) and 80% (16/20), respectively. In both treated and control animals these discolorations were qualitatively similar; none were considered to be unique. In the opinion of the pathologist, these various discolorations of the lungs were not considered to be of toxicological significance with respect to the test article.
Other postmortem findings either occurred in the treated and control animals with comparable incidences and severities or they occurred sporadically. They were not considered to be related to the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No aborted pregnancies were noted during the study.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea and mean number of uterine implantations per pregnant female were comparable between the control and treated groups. The mean pre-implantation loss index was considered comparable between these same groups. A slight increase in the mean pre-implantation loss index was seen in the mid-dose group as compared to the control group; however, this difference from control was not statistically significant and in the absence of a similar increase in the high-dose group, was not considered indicative of a treatment-related effect.
No adverse effect of treatment was evident from uterine implantation data.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of resorption sites, the mean ratio of resorptions to implants and the incidence of females with at least one resorption site in utero comparable between the control and each treated group.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of live fetuses per pregnant female was comparable between the control and treated groups. The only dead fetus recovered in utero was from the litter of control.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Premature delivery of a litter during the Day 26-30 gestation interval was seen for two control females (10.0%), one low-dose female (5.0%), two mid-dose females (10.0%) and one high-dose female (5.3%). These incidences were comparable between the control and treated groups and no adverse effect of treatment was indicated.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rates were comparable between the control and treated groups. The pregnancy rate was 100% in the control, low and mid-dose groups and 95% (19/ 20 ) in the high-dose group. A total of 72 females (18 control, 19 low-dose, 17 mid-dose and 18 high-dose females) survived to scheduled sacrifice (Day 30 of gestation). Only one of these females (high-dose female) did not have uterine implantations at sacrifice and no foci were visualized following uterine staining. This was the only non-pregnant animal on study.

Other effects:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal weights distinguished by sex and as a composite for both sexes, were slightly lower than control in each of the treated groups but these differences from control data were not statistically significant. Mean fetal weight data for the treated groups were within the range of recent historical control data for this laboratory (Males: mean = 46.7 g; range = 40.0 to 50.4 g. Females: mean = 45.5 g; range = 39.6 to 48.1 g. Both: mean = 46.3 g; range = 40.5 to 49.6 g).
No adverse effect of treatment was evident from fetal weight data.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean number of male and female fetuses per litter and the ratio of male to female fetuses were comparable between the control and treated groups.
No adverse effect of treatment was evident from fetal sex distribution data.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with external malformations for the control and low-dose groups were 0.6% (1/173 fetuses) and 0.6% (1/170 fetuses), respectively, No external malformations were seen in the 156 mid-dose fetuses (17 litters) or 158 high-dose fetuses (17 litters) evaluated. The following external malformations were seen during this study: distended abdomen in one fetus from the litter of control female; and the presence of a single external nare and absence of upper incisors in one fetus from the litter of low-dose female.
No external malformations were seen among the pups/fetuses recovered from females that delivered prematurely. A total of 39 pups were examined externally [14 control pups from two litters, six low-dose pups from a single litter and 19 mid-dose pups /fetuses from two litters].
No adverse effect of treatment was evident from the fetal external examination data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with skeletal malformations for the control, low-, mid- and high-dose groups were 5.2% (9/173 fetuses), 8.9% (15/169 fetuses), 5.8% (9/156 fetuses) and 3.8% (6/158 fetuses), respectively. The incidences of litters containing fetuses with skeletal malformations for these same groups were 44.4% (8/18 litters), 47.4% (9/19 litters), 52.9% (9/17 litters) and 29.4% (5/17 litters), respectively. The incidences of litters containing fetuses with skeletal malformations did not differ statistically between the control and treated groups and were considered comparable.
Fused sternebrae, a minor skeletal malformation, was the most common malformation seen during the study. The incidences of fetuses with fused sternebrae for the control, low-, mid- and high-dose groups were 2.3% (4/173), 3.6% (6/169), 0.6% (1/156) and 3.2% (5/158), respectively. The incidences of litters containing fetuses with fused sternebrae for the control, low-, mid- and high-dose groups were 22.2% (4/18), 10.5% (2/19), 5.9% (1/17) and 23.5% (4/17), respectively. The incidence of fused sternebrae both on a per fetus and per litter basis for this study was considered similar between the control and treated groups and within the range of recent historical control data.
Angulated arch (es) of the hyoid, also considered a minor skeletal malformation, was seen at low incidence in the control, low- and mid-dose groups; this malformation was not seen among the 158 high-dose fetuses. The incidences of fetuses with angulated arch (es) of the hyoids for the control, low- and mid-dose groups were 2.9% (5/173), 2.4% (4/169) and 1.9% (3/156), respectively. These incidences were similar between the control and treated groups and were within the range of recent historical control data.
Excluding those fetuses with only minor skeletal malformations as presented above, the incidences of fetuses with other skeletal malformations for the control, low- , mid- and high-dose groups were 1.2% (2/173), 3.0% (5/169), 3.2% (5/156) and 0.6% (1/158), respectively. Dissimilar malformations of the cranial bones (frontals , nasals) were seen in three fetuses (one fetus from each of the control, low- and mid-dose groups) that had either external or visceral malformations.
One fetus from the litter of control female had misshapen nasals and fused frontal bones; this fetus was noted during the visceral examination with defects of the aortic arch and a cardiac malformation. One fetus from the litter of low-dose female had fused frontal bones, nasals fused and misshapen and fused premaxillary bones; this fetus was noted externally with a single external nare. One fetus from the litter of mid-dose female had fused frontal bones and at visceral examination this fetus was noted with ectopic kidneys.
The only other malformation seen with some frequency during the study involved fused/branched ribs with or without vertebral involvement. Fused/branched ribs were seen in one control fetus (0.6%), one low-dose fetus (0.6%) and two mid-dose fetuses from different litters (1.3%). The incidence of litters containing fetuses with skeletal malformations was similar for all study groups (control and treated) ; most of these observations involved minor skeletal malformations (fused sternebrae , angulated arch of the hyoid) which were seen with similar incidence among the control and treated groups.
Other skeletal malformations seen during the study among the treated groups , occurred in low incidence, were dissimilar in nature and showed no dose-related trends. Thus, no adverse effect of treatment was indicated from skeletal malformation data.
No skeletal malformations were seen in the 39 pups/fetuses recovered from females that delivered prematurely (14 control , 6 low-dose and 19 mid-dose pups/fetuses were evaluated).
The incidences of fetuses with at least one ossification variation for the control, low-, mid- and high-dose groups were 64.2% (111/173), 52.7% (89/169), 66.0% (103/156) and 62.7% (99/158), respectively. The incidence of litters containing at least one fetus with an ossification variation was comparable between the control and treated groups; this was 94.4% for the control group and 100% for each of the treated groups. No adverse effect of treatment was evident from ossification variation data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with visceral malformations for the control, low-, mid- and high-dose groups were 1.2% (2/173), 0.6% (1/170), 1.3% (2/156) and 1.3% (2/158), respectively. The incidences of litters containing fetuses with visceral malformations for the control, low-, mid- and high-dose groups were 11.1% (2/18), 5.3% (1/19), 11.8% (2/17) and 5.9% (1/17), respectively and these incidences were comparable between the control and each treated group.
In the control group, one fetus from the litter of female had persistent truncus arteriosus (a defect of the aortic and pulmonary arches) and interventricular septal defect. Ascites (fluid in the abdominal cavity) and an enlarged heart were seen in the one control fetus noted externally with a distended abdomen.
In the low-dose group, the only visceral malformation noted was slight distention of the lateral ventricles of the brain and this malformation was seen in one fetus from the litter of female. Distended renal pelvis (without a renal papillae present) was seen in one fetus from the litter of mid-dose female and two fetuses from the litter of high-dose female. The only other visceral malformation seen during the study was ectopic kidney seen in one fetus from the litter of mid-dose female. Several of these malformations have been noted at low incidence in historical control data for our laboratory.
No adverse effect of treatment was evident from the visceral malformation data.

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

>= 20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1. Overview of study results

Dose (mg/kg bw/day)	0	5	10	20	dr
Maternal effects
Mortality	-	-	1a	1a	-
Clinical signs and	-	-	-	-	-
behaviour	-	-	-	-	-
Pregnant animals	20/20	20/20	20/20	20/20	-
Abortions	-	-	-	-	-
Early delivery	2	1	2	1	-
Females with viable foetuses at C-section	18	19	17	17	-
Body-weight gain
- day 7-10	6	8	-9	-26	dr
- day 10-13	24	3	22	8
- day 13-16	86	78	53	21	dr
- day 16-19	21	22	6	-9	dr
- day 19-30	94	38	75	111
- day 7-19	136	111	71	-8*	dr
Food consumption
- day 7-8	47	45	42	40*b
Pathology	No treatment related findings				-
Litter response
Live foetuses	No treatment related findings				-
Fetal weight	No treatment related findings				-
Post implantation loss	No treatment related findings				-
Sex ratio	No treatment related findings				-
Examination of foetuses
External observations	No treatment related findings				-
Skeletal findings	No treatment related findings				-
Visceral findings	No treatment related findings				-

dc/ic: statistically significantly decreased/increased compared to the controls

d/i: decreased/increased, but not statistically significantly compared to the controls

dr: dose-related

+/-: increase/decrease without statistical analysis

a: The female that died in the middle dose group had fluid in the lungs and trachea, both suggestive of pulmonary oedema. No indications on the cause of death of the other female were found.

b: The mean food intake in high dosed females during the treatment period differed from controls ranging from -8.7% at day 10 to -35.0% at day 17.

* p<0.05, ** p<0.01

 

Table 2. Reproductive performance parameters

Observation	Dose level (mg/kg bw/day)
	0 (control)	5	10	20
Animals Assigned (Mated)	20	20	20	20
Animals Pregnant	20	20	20	19
Animals Non-pregnant	0	0	0	1
Animals aborted	0	0	0	0
Premature births	2	1	2	1
Litters with viable foetuses	18	19	17	17
Litters with all resorptions	0	0	0	0
Mortality	0	0	1	1
Mean no corpora lutea/dam	11.0	10.1	11.2	10.0
Mean no implants/dam	10.2	9.5	9.5	9.9
Mean pre-implantation loss (%)	0.077	0.063	0.153	0.061
Number viable foetuses	172	170	156	158
Mean litter size	9.6	8.9	9.2	9.3
Mean number of males/litter	4.0	3.9	4.6	4.5
Mean number of females/litter	4.0	5.1	4.6	4.8
Dead foetuses	1	0	0	0
Number of resorptions	11	10	6	11
Mean number resorptions	0.6	0.5	0.4	0.6
Mean number resorptions/implants	0.054	0.0449	0.031	0.067
Litters with resorptions	7	2	4	10
Mean foetal weight – viable foetuses (g)	45.60	43.30	43.32	42.91
Mean male foetal weight (g)	46.88	43.19	43.34	43.09
Mean female foetal weight (g)	44.93	43.34	41.99	41.00
Ratio of viable foetuses (total males/total females)	1.0	0.8	1.0	1.0

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, the NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects.

Executive summary:

In this study (83-3 under GLP), the test substance was tested for its embryotoxic, fetotoxic, and teratogenic potential in rabbits. Test article was suspended in 0.5% (w/v) aqueous methyl cellulose vehicle and administered by gastric intubation as a single dose daily during the Day 7-19 gestation interval. Dose levels evaluated were 5, 10 and 20 mg/kg bw/day. Each test group contained 20 mated female New Zealand White rabbits. Included in the study was a vehicle-treated control group which also contained 20 mated females. All animals were observed twice daily (morning, afternoon) for obvious toxicological effects and mortality. Each animal was weighed and given a detailed physical examination on Days 0, 3, 7, 10, 13, 16, 19, 24 and 30 of gestation. Food consumption was recorded on Days 1 , 3, 7, 10, 13, 16, 19, 24 and 29 of gestation. Animals aborting or delivering prematurely were killed on the day such evidence was observed and given a gross postmortem evaluation. Intact pups/fetuses recovered from females that delivered prematurely (Days 26-30 of gestation) were evaluated for external malformations, eviscerated (gross evaluation of viscera) and evaluated for skeletal malformations following processing for Alizarin Red S staining of the ossified structures. Surviving females were killed on Day 30 of gestation and given a gross postmortem examination. The gravid uterus was removed, weighed and evaluated for the presence of live, dead and resorbed fetuses. Fetuses recovered in utero were weighed and given a gross external examination. Subsequently, each fetus was processed for visceral evaluation (fresh microdissection procedure ) and skeletal evaluation (Alizarin Red S stained specimens). The number of corpora lutea on each ovary was also recorded for each female.

No mortality occurred in the control or low-dose group. In the control group, two females delivered prematurely and the remaining 18 females survived to scheduled sacrifice on Day 30 of gestation. In the low-dose group (5 mg/kg/day), one female delivered prematurely and the remaining 19 females survived to Day 30 scheduled sacrifice. The pregnancy rate for the control and low-dose group was 100%. No maternal toxic, embryotoxic, fetotoxic or teratogenic effects were evident at the 5 mg/kg bw/day dose level. In the 10 mg/kg bw/day dose group, one female died on Day 10 of gestation after receiving three days of treatment. At gross postmortem examination, this female was noted with fluid in the lungs and frothy fluid in the trachea, both findings are suggestive of a pulmonary oedema which may or may not have resulted from an intubation error. Two mid-dose females delivered prematurely and the remaining 17 females in this group survived to Day 30 scheduled sacrifice. Pregnancy rate for the mid-dose group was 100%. The 10 mg/kg bw/day dose level was not considered to be maternally toxic, embryotoxic, fetotoxic or teratogenic. In the 20 mg/ kg/day dose group, one pregnant female died, one female delivered prematurely and the remaining 18 females survived to scheduled sacrifice. Seventeen of these eighteen high-dose females were pregnant as evidenced by the presence of uterine implantation sites (pregnancy rate = 95%). One high-dose female died on Day 13 of gestation after six days of treatment. The cause of death of this female could not be determined on the basis of postmortem findings. High-dose females experienced a mean weight loss during the treatment period (Days 7-19) in comparison to a mean weight gain in the control group over this same interval and these data differed statistically between these two groups. Mean food consumption for the high-dose group during the treatment period (Days 7, 10, 13, 16 and 19) was also lower than control data; however, only on Day 7 was this difference from control statistically significant. No other maternal toxicity was evident at the 20 mg/ kg/day dose level and this dose level was not considered to be embryotoxic, fetotoxic or teratogenic.

In conclusion, the NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects.