Ethanone, 2,2-dichloro-1-[5-(2-furanyl)-2,2-dimethyl-3-oxazolidinyl]-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June - July 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April – Mai 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

; the respective study serves for the purpose of a range-finding study in rabbits.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

November 1984

Deviations:

yes

Remarks:

; eight instead of twenty inseminated females were used in the range-finding study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 12, 1981

Deviations:

yes

Remarks:

; eight instead of twenty inseminated females were used in the range-finding study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

For the purposes of dose calculation the test material was considered to be 100%.
- Final test material formulation: suspension

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Hazleton Research Products, Inc., Denver, Pennsylvania,
- Age upon receipt: 5 months
- Weight at study initiation: 3440-4292 g
- Fasting period before study: restricted to approx. 150 g/animal/day during acclimation period
- Housing: individually in clean, stainless-steel wire bottom cages suspended above cage board
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67-71
- Humidity (%): 38-94%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare each liter of the vehicle (0.5 % aqueous methylcellulose), 1000 ml of deionized water was heated to approximately 70°C and 5.0 g of the control material powder was gradually added; the mixture was stirred until clear. The vehicle was prepared prior to study initiation and stored refrigerated between periods of use. An appropriate amount of the test material was weighed for each group into a tared weigh boat. The test material was transferred to a mortar, ground to a fine powder and triturated with the vehicle (0.5 % methylcellulose) until a slurry was formed. This mixture was transferred to a graduated cylinder via vehicle rinses and was then brought to the appropriate volume with the vehicle. The graduated cylinder was inverted several times, then the dosing preparation was transferred to a storage container. The graduated cylinder was rinsed with an additional 1500 ml of the vehicle, which was then added to the storage container to attain the appropriate concentration for each dose group. The preparation was mixed on a Polytron PT6000 for approximately five minutes to reduce particle size. A magnetic stir bar was added and the mixture was stirred continuously throughout the sampling and dosing procedures and between periods of use. The dosing preparations were visually inspected for homogeneity prior to dispensation for administration. Dosing preparations were prepared once during the treatment period (April 29, 1992) and were stored in sealed containers at room temperature between periods of use.

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 5 ml/kg was used for all dose levels

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected from test material dosing preparations formulated on April 29, 1992 and analyzed for stability and concentration. Duplicate samples (15 mL) were collected from the middle of each dosing preparation. Concentration was verified on one set of samples for all dose groups, included the control group, on April 30, 1992. Stability was verified on the second set of samples for the low and high dose groups on the last day of dosing (May 16, 1992). The methology and results of these analyses are presented in Appendix B of the report. The dosing preparations for all dose groups contained the amounts of test material specified in the protocol. The dosing preparations were homogeneous and stable for the duration of the dosing period.

Details on mating procedure:

Impregnation procedure: artificial insemination (the day of insemination was designated gestation day 0.)

Duration of treatment / exposure:

Gestation Days 7 through 19 (13 consecutive days)

Frequency of treatment:

once a day

Duration of test:

30 days (from the day of artificial insemination up to the last Cesarean section)

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day

No. of animals per sex per dose:

8 females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a preliminary 5-day toxicity study using non-mated females (WIL-50207A; Appendix A of Report).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily (for mortality, moribundity, general appearance and behaviour)

DETAILED CLINICAL OBSERVATIONS: Yes, daily (individual detailed clinical observations were recorded daily from gestation days 0 through 29 prior to dosing during the treatment period and approximately one hour following dosing)

BODY WEIGHT: Yes, on gestation days 0, 7, 10, 13, 19, 24, and 29
Maternal body weights were recorded indivudually. A group mean was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-19, 19-29 and 0-29.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Females which aborted during the experimental period were necropsied that day, the other females were necropsied on Gestation Day 29, the scheduled day of Cesarean Section.
- Organs weighed: The liver, kidneys and spleen from each dam were excised, trimmed, weighed and the findings recorded. The uteri and ovaries were excised and the trimmed uterus with contents was then weighed.
- Organs examined histopathologically: Maternal tissues were retained in 10 % neutral buffered formalin for possible future histopathological examination only as indicated by the gross findings. No histopathological examinations were performed.

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, incl. Salewski staining
- Number of late resorptions: Yes
The location of all fetuses, early and late resorptions in the uterus was recorded.

Fetal examinations:

Recognizable fetuses from dams which delivered prior to gestation day 29 were examined externally and preserved in 10 % neutral buffered formalin, only if malformations were noted. Fetal findings for females which aborted were not included in any tabulation or statistical analyses. Recognizable fetuses from dams which delivered on gestation day 29 were examined as described for those animals surviving to the scheduled necropsy.

A detailed examination of each fetus was conducted to include, but was not limited to the eyes, palate, and external orifices. Findings were recorded as either developmental variations or malformations. Each fetus was weighed euthanized and discarded. Crown-rump measurements were recorded for late resorption and the tissues were discarded.

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

Statistics:

- Fisher's Exact test
- Mann-Whitney U-test
- One-way ANOVA with Dunnett’s test

Postimplantation loss, live litter size, fetal body weights and numbers of corpora lutea and implantation sites were assessed as appropriate, on a group mean basis or as a proportional (%) litter comparison.

Indices:

Group mean litter basis: postimplantation loss / litter = numer of dead fetuses, resorptions/group per number of gravid females/group

Proportional litter basis: summation per group (%) = postimplantation loss/litter (%)* per number of litters/group
* = number of dead fetuses, resorptions/litter x 100 per number of implantation sites/litter

Historical control data:

Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The predominant treatment-related clinical signs were excreta-related (decreased defecation, feces appear small in size and dark yellow urine). Decreased defecation and small feces were noted in all dose groups, including the control group. However, the increased frequency of these findings in the 50, 100 and 175 mg/kg/day groups in a dose-related manner was indicative of a treatment-related effect. Dark yellow urine was only observed in animals at dose levels of 100 and 175 mg/kg/day and, therefore, was apparently related to compound administration.
The only other clinical sign that could possibly be related to compound administration was dried brown matting on the anogenital area which was slightly increased in the 175 mg/kg/day group.
Other clinical signs in the treated groups occurred infrequently and/or did not occur in a dose-related manner.
[cp. tables 2 and 3 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Mortality:

no mortality observed

Description (incidence):

The females that aborted (five at 175 mg/kg/day, one at 100 mg/kg/day and one at 50 mg/kg/day) were sacrificed the day they aborted. Spontaneous mortality was not observed.
[cp. table 1 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose-related group mean body weight losses occurred in all treated groups during the overall treatment period (gestation days 7-19). These losses were statistically significant (p<0.05 or p<0.01) in the 50, 100 and 175 mg/kg/day groups when compared to the control group value.
Group mean body weight losses or no change in body weight were recorded in the 50, 100 and 175 mg/kg/day groups for all intervals during the treatment period (gestation days 7-10, 10-13 and 13-19). During gestation days 7-10 in the 100 and 175 mg/kg/day groups and gestation days 10-13 in the 175 mg/kg/day group, the losses for these groups were statistically significant (p<0.01). During the overall post-treatment period (gestation days 19-29), group mean body weight gains in the 50, 100 and 175 mg/kg/day groups were increased when compared to the control group; the values in the 50 and 100 mg/kg/day groups were statistically significant (p<0.01). Group mean body weights at dose levels of 100 and 175 mg/kg/day were reduced on gestations days 10, 13, 19, 24 and 29, and were statistically significant (p<0.05) on gestation days 19 and 24 in the 100 and 175 mg/kg/day groups, respectively. Group mean body weights in the 50 mg/kg/day group were generally comparable to the control group throughout the study.
At a dose level of 10 mg/kg/day, a slightly reduced group mean body weight gain on gestation days 7-10 and a group mean body weight loss during gestation days 13-19 (but not statistically significant) were recorded. Group mean body weight gain at this same dose level was comparable to the control group during gestation days 10-13. For the overall post-treatment period (gestation days 19-29), group mean body weight gains in the 10 mg/kg/day group were increased and statistically significant (p < 0.01) when compared to the control group. Group mean body weights in this same dose group were comparable to the control group throughout the study.
Gravid uterine weights were reduced in a dose-related manner in the 100 and 175 mg/kg/day groups due to increases in postimplantation loss at these same dose levels; the gravid uterine weight in the 175 mg/kg/day group represented only one gravid female with an entirely resorbed litter. Group mean net body weights and net body weight changes in the treated groups were similar to the control group.
[cp. tables 4, 5 and 6 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney, liver and spleen weights in the single surviving 175 mg/kg/day group gravid female were increased relative to the control group. At a dose level of 100 mg/kg/day, the mean liver weight was increased and statistically significant (p<0.05) relative to the control group. Mean liver weight was also slightly increased, but not statistically significant, in the 50 mg/kg/day group compared to the control group. Mean kidney and spleen weights in the 50 and 100 mg/kg/day group were comparable to the control group. All group mean organ weights in the 10 mg/kg/day group were
comparable to the control group values.
[cp. table 7 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings observed in surviving animals on gestation day 29 were limited and apparently unrelated to test material administration.

Necropsy findings in the animals that aborted occurred infrequently and were apparently unrelated to treatment.
[cp. table 18 in WI-92-154_Individual Data in block "Overall remarks, attachments"]

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Five animals in the 175 mg/kg/day group (nos. 13646, 13663, 13667, 13669 and 13675) aborted, one each on gestation days 20, 23, and 24 and two on gestation day 28; these abortions were considered treatment-related. One 100 mg/kg/day group female (no. 13681) aborted on gestation day 28, and one 50 mg/kg/day group female (no. 13679) aborted on gestation day 29. In the WIL historical control data, 23 of 53 data sets dosed orally (43 %) had at least one female which aborted. However, due to group mean body weight losses in the 50 and 100 mg/kg/day groups for the overall dosing interval, these abortions were considered to be possible effects of treatment. All other animals survived to the scheduled necropsy on gestation day 29.
[cp. table 1 in WI-92-154_Summary Tables, WI-92-154_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

When evaluated on a proportional basis, the values for postimplantation loss were increased in the 100 and 175 mg/kg/day groups (43.3% and 100.0%, respectively) relative to the control group value (12.8%). These increases were considered to be treatment-related.

The numbers of implantation sites and corpora lutea at dose levels of 100 and 175 mg/kg/day were comparable to the control group values.

[cp. tables 8 and 9 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One female in each of the dose groups 100 and 175 mg/kg/day had completely resorbed litters (primarily early resorptions). The female in the 175 mg/kg/day group (no. 13647) with the entirely resorbed litter was the only surviving gravid animal at this dose level.
[cp. tables 8 and 9 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The number of late resorptions in the 50 mg/kg/day group (1.3/litter) was increased compared to the control group (0.1/litter). Due to the lack of a similar response in the higher dose groups, however, no relationship to treatment was evident.
[cp. tables 8 and 9 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Dose descriptor:

LOEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

number of abortions

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were inadvertently not recorded for 50 mg/kg/day group animal no. 13679 which delivered on gestation day 29.

The mean fetal body weight at a dose level of 100 mg/kg/day was comparable to the control group. No adverse effects on intrauterine growth or survival were apparent at dose levels of 10 and 50 mg/kg/day.
[cp. tables 8 and 9 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The mean number of viable fetuses at dose levels of 100 and 175 mg/kg/day (3.5 and 0 fetuses/litter, respectively) was decreased when compared to the control group value (5.9 fetuses/litter) in a dose related manner.
[cp. tables 8 and 9 in WI-92-154_Summary Tables and also WI-92-154_Individual Data in block "Overall remarks, attachments"]

Changes in sex ratio:

not specified

External malformations:

effects observed, treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for fetal morphological examination were 47(8), 50(8), 47(7), 14(4) and 0(0) in the control, 10, 50, 100 and 175 mg/kg/day groups, respectively.

Malformations were observed in 0(0), 0(0), 6(4), 5(2) and 0(0) fetuses (litters) in these same dose groups, respectively. In the 175 mg/kg/day group, the absence of viable fetuses precluded any fetal morphological evaluation. The number of litters with external malformations in the 50 mg/kg/day group was increased and statistically significant (p<0.05).

Hydrocephaly (with or without a dome-shaped head) was observed in 5(3) and 3(1) fetuses (litters) in the 50 and 100 mg/kg/day groups, respectively. The percentages of fetuses (litters) affected in these dose groups [10.6 % (42.9 %) and 21.4 % (25.0 %), respectively] exceeded the maximum value in the WIL historical control data [3.0% (5.6%)].

Multiple anomalies were observed in five of these fetuses with hydrocephaly (nos. 13636-4 and -5 in the 50 mg/kg/day group and nos. 13686-4, -5 and -6 in the 100 mg/kg/day group). Fetus nos. 13636-4 and -5 also had cleft palate (entire length). Fetus nos. 13686-4, -5 and -6 also had open eyelids (unilateral or bilateral).
The remaining external malformations included multiple subcutaneous hemorrhages in 50 mg/kg/day group fetus no. 13685-9, an omphalocele in 100 mg/kg/day group fetus no. 13651-1 and an open eyelid in 100 mg/kg/day group fetus no. 13686-3.

[cp. tables 10 and 11 in WI-92-154_Summary Tables, WI-92-154_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Multiple anomalies were observed in five of these fetuses with hydrocephaly (nos. 13636-4 and -5 in the 50 mg/kg/day group and nos. 13686-4, -5 and -6 in the 100 mg/kg/day group). Fetus nos. 13636-4 and -5 also had carpal and/ or tarsal flexure (bilateral), the pelvic girdle was more narrow than normal and the pectoral girdle was more narrow than normal (the forelimbs were located more ventral and medial than normal). Fetus no. 13636-4 additionally had a stemebra more prominent than normal.
In the 100 mg/kg/day group fetuses with multiple anomalies, fetus nos. 13686-4 and -6 also had carpal flexure (bilateral) and the pectoral girdle was more narrow than normal (the forelimbs were located more ventral and medial than normal).

The percentage of fetuses (litters) with carpal and/or tarsal flexure in the 50 and 100 mg/kg/day groups [4.3% (14.3%) and 14.3% (25.0%)] exceeded the maximum value in the WIL historical control data [1.7 % (7.1 %)]. The other anomalies in these fetuses were not previously observed in the historical control data set.

[cp. tables 10 and 11 in WI-92-154_Summary Tables, WI-92-154_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Conclusions:

In conclusion, maternal toxicity was expressed at a dose level of 175 mg/kg/day by five abortions. Maternal toxicity was also expressed at dose levels of 100 and 175 mg/kg/day by changes in the general clinical condition of the animals, the inhibition of body weight gains and increases in organ weights. The single abortion at a dose level of 100 mg/kg/day was another possible expression of maternal toxicity. Maternal toxicity was expressed at a dose level of 50 mg/kg/day by changes in the general clinical condition of the animals and a slight increase in mean liver weight. Inhibited body weight gains and one abortion in the 50 mg/kg/day group were considered to be possible expressions of maternal toxicity. In the 10 mg/kg/day group, inhibited body weight gains were possible expressions of maternal toxicity. Developmental toxicity was expressed in the 100 and 175 mg/kg/day groups by increases in postimplantation loss (43.3% and 100.0%, respectively). Developmental toxicity was also expressed in the 50 and 100 mg/kg/day groups by several malformations (primarily hydrocephaly). Based on the results of this study, dose levels of 2, 10 and 50 mg/kg/ day were selected for the developmental toxicity study of the test substance in rabbits.

Executive summary:

This study was conducted to obtain a preliminary assessment of the potential of the substance to induce maternal and embryo/fetal toxicity in rabbits.

The test material was mixed with 0.5% aqueous methylcellulose and administered daily by gavage (5 mL/kg) to 5 groups of 8 artificially inseminated New Zealand White rabbits during gestation days 7 through 19. Dose levels were 0, 10, 50, 100, and 175 mg/kg/day. All dosing solutions were prepared once at the beginning of the study and stored at room temperature until used. The concentration of test article in all dosing suspensions was confirmed prior to administration of the first dose and stability was verified by analyzing the lowest and highest concentrations on the last day of dosing. Food and water were available ad libitum. The animals were observed at least twice daily for mortality and gross signs of toxicity. Detailed clinical observations were recorded daily. Body weights were recorded on gestation days 0, 7, 10, 13, 19, 24, and 29. Gross necropsies were conducted on all animals that died during the study or that were sacrificed after aborting. All surviving animals were sacrificed on gestation day 29. Gross postmortem examinations were conducted and liver, kidneys, spleen and uterus weights were determined. The uterus and ovaries were examined, and the number and location of viable and nonviable fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Fetuses were weighed and examined for external malformations and variations. Crown-rump lengths were recorded for late resorptions.

Analysis of the dosing suspensions confirmed that all group means were within 15% of target concentration. Homogeneity and stability was also verified. There were no maternal deaths at any treatment level. Abortions occurred in the 50, 100 and 175 mg/kg dose groups (1/8, 1/6 and 5/6 gravid females, respectively) but there were none in the control or low-dose (10 mg/kg/day) groups. Clinical observations associated with treatment at the 50, 100 and/or 175 mg/kg/day dose level included diarrhea, decreased defecation, decreased urination, and urine that appeared dark yellow. An increased incidence of mucoid feces was observed in the 175 mg/kg/day group. Mean weight loss was noted during the treatment period (days 7-19) in the 50, 100 and 175 mg/kg/day groups, while these groups showed increased weight gains in the post-treatment period. In the lowest dose group, slightly reduced weight gain or weight loss during the treatment period and increased weight gain in the post-treatment period were also considered possible treatment-related effects. The mean liver weight in the 100 mg/kg/day group was significantly greater than control (37%), and liver, kidney and spleen weights in the surviving female from the highest dose group were greater than control. The mean liver weight in the 50 mg/kg/day dose was also increased (approximately 15%) but the difference from control was not significant. There were no viable fetuses in the 175 mg/kg/day group. Postimplantation loss (%) was increased and the mean number of live fetuses/litter was decreased in the 100 mg/kg/day group. There were no adverse effects on intrauterine growth or survival at the 10 or 50 mg/kg/day dose levels and no effects on fetal weights in any dose group. Malformations ( eg: hydrocephaly, cleft palate, malpositioned limbs/abnormal flexure of the paws, narrow pectoral or pel vie girdle, omphalocele, open eyes and hemorrhage) were observed, in fetuses from 4 litters of the 50 mg/kg/day group and 2 litters from the 100 mg/kg/day group.

In conclusion, administration of the formulated test material to pregnant rabbits resulted in maternal toxicity at the three highest dose levels tested, 50, 100 and 175 mg/kg/day. Slight evidence of maternal toxicity was noted at the 10 mg/kg/day group. Developmental toxicity (increased postimplantation loss and malformations) was seen in the 50, 100 and 175 mg/kg/day dose groups.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

November 1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 12, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2-Dichloro-1-[5-(2-furyl)-2,2-dimethyl-1,3-oxazolidin-3-yl]ethanone

EC Number:

601-807-6

Cas Number:

121776-33-8

Molecular formula:

C11H13CL2NO3

IUPAC Name:

2,2-Dichloro-1-[5-(2-furyl)-2,2-dimethyl-1,3-oxazolidin-3-yl]ethanone

Test material form:

solid

Specific details on test material used for the study:

For the purposes of dose calculation the test material was considered to be 100%.
- Final test material formulation: suspension

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Hazleton Research Products, Inc., Denver, Pennsylvania,
- Age upon receipt: approx. 7 months
- Weight at study initiation: 3349-4563 g
- Fasting period before study: restricted to approx. 150 g/animal/day during acclimation period
- Housing: individually in clean, stainless-steel wire bottom cages suspended above ground corn cob bedding
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: six weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68 - 72
- Humidity (%): 54-90%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare each liter of the vehicle (0.5 % aqueous methylcellulose), 1000 ml of deionized water was heated to approximately 70°C and 5.0 g of the control material powder was gradually added; the mixture was stirred until clear. The vehicle was prepared prior to study initiation and stored refrigerated between periods of use. An appropriate amount of the vehicle (3500 ml) was dispensed into a storage container. A magnetic stir bar was added and the vehicle was stirred continuously during the dosing procedure. An appropriate amount of the test material was weighed for each group into a tared weigh boat. The test material was transferred to a mortar, ground to a fine powder and triturated with the vehicle (0.5 % methylcellulose) until a slurry was formed. This mixture was transferred to a graduated cylinder via vehicle rinses and was then brought to volume (2000ml) with the vehicle. The graduated cylinder was inverted several times, then the dosing preparation was transferred to a storage container. The graduated cylinder was rinsed with an additional 1500 ml of the vehicle, which was then added to the storage container to attain a total volume of 3500 ml. The preparation was mixed on a Polytron PT6000 homogenizer for approximately five minutes to reduce particle size. A magnetic stir bar was added and the mixture was stirred continuously throughout the sampling and dosing procedures. An adequate amount of the preparations for each group was dispensed daily for dosing. Dosing preparations were made twice during the treatment period (June 9 and June 18, 1992) and were stored at room temperature.

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 5 ml/kg was used for all dose levels

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test material preparations were analyzed at WL Researchbboratories, Inc. Samples were drawn from all groups, including the control group, from the first batch preparation to verify concentration. Homogeneity was verifed in the low and high dose groups only. Samples were drawn from each dose group from the second batch preparation to verify concentration.Concentration analyses for the high dose group were repeated twice due to apparently unrepresentative aliquot samples. On the last day of dosing, aliquots were taken from the low and high dose group preparations and analyzed for stability. The dosing preparations were homogeneous, contained the designated amount of test material specified in the protocol and were stable for the duration of dosing.

Details on mating procedure:

- Impregnation procedure: artificial insemination (the day of insemination was designated gestation day 0.)

Duration of treatment / exposure:

during gestation days 7 through 19 (13 consecutive days)

Frequency of treatment:

once a day

Duration of test:

31 days (from the day of artificial insemination up to the last laparohysterectomy)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

20 females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a preliminary range-finding study with the substance (WIL-50207; linked per cross-reference to this study record)

As conclusion it is stated in the study report of the range-finding study: "maternal toxicity was expressed at a dose level of 175 mg/kg/day by five abortions. Maternal toxicity was also expressed at dose levels of 100 and 175 mg/kg/day by changes in the general clinical condition of the animals, the inhibition of body weight gains and increases in organ weights. The single abortion at a dose level of 100 mg/kg/day was another possible expression of maternal toxicity. Maternal toxicity was expressed at a dose level of 50 mg/kg/day by changes in the general clinical condition of the animals and a slight increase in mean liver weight. Inhibited body weight gains and one abortion in the 50 mg/kg/day group were considered to be possible expressions of maternal toxicity. In the 10 mg/kg/day group, inhibited body weight gains were possible expressions of maternal toxicity. Developmental toxicity was expressed in the 100 and 175 mg/kg/day groups by increases in postimplantation loss (43.3% and 100.0%, respectively). Developmental toxicity was also expressed in the 50 and 100 mg/kg/day groups by several malformations (primarily hydrocephaly). Based on the results of this study, dose levels of 2, 10 and 50 mg/kg/ day were selected for the developmental toxicity study of the test substance in rabbits."
The guideline used (EPA OPP 83-3, 1984 or OECD TG 414, 1981) states that the highest dosage level should ideally induce some overt maternal toxicity such as slight weight loss, but not more than 10 per cent maternal deaths. Thus, taking into account especially the above mentioned effects on the body weight gains, where effects were seen up to the lowest dose (10 mg/kg/day) this criteria is considered to be fullfilled.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily (for mortality, moribundity, general appearance and
behaviour)

DETAILED CLINICAL OBSERVATIONS: Yes, daily (individual detailed clinical observations were
recorded daily from gestation days 0 through 29 prior to compound administration during the dosing period and approximately one hour following dosing)

BODY WEIGHT: Yes, on gestation days 0, 7, 10, 13, 19, 24, and 29
Maternal body weights were recorded individually. A group mean was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-19, 19-29 and 0-29

FOOD CONSUMPTION: Yes, during days 0-29
Individual food consumption was recorded on days 0 through 29 of gestation. Food intake was calculated as g/animal/day and g/kg/day for corresponding weight change intervals. On the occasions when food intake could not be measured for one of the days in a given interval, food intake was calculated using the appropriate number of days for that interval.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- A gross necropsy was performed on females which died during the course of the study. Females which aborted during the experimental period were necropsied that day, the other females were necropsied on Gestation Day 29, the day of scheduled necropsy.
- Organs weighed: The liver, kidneys and spleen from each dam were excised, trimmed, weighed and all findings recorded. The uteri and ovaries were excised and the trimmed uterus with contents was then weighed.
- Organs examined histipathologically: The livers and maternal tissues from all animals were preserved in 10 % neutral buffered formalin. The livers from control and high-dose animals were examined histopathologically.

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, incl. Salewski staining
- Number of late resorptions: Yes
The location of all fetuses, early and late resorptions in the uterus was recorded.

Fetal examinations:

Recognizable fetuses from dams which delivered prior to gestation day 29 were examined externally and preserved in 10 % neutral buffered formalin. Intrauterine findings from dams which aborted were not included in any tabulation or statistical analyses with data from dams surviving to termination. Recognizable fetuses from dams which delivered on gestation day 29 were examined as described for those animals surviving to the scheduled necropsy and the findings were presented separately.

Each fetus was individually weighed. A detailed external examination of each fetus was conducted to include, but was not limited to, the eyes, palate and external orifices and each finding was recorded. Crown-rump measurements were recorded for late resorptions and the tissues were discarded. The sex of each fetus was determined by an internal examination. Each fetus was examined viscerally by a modification of the Stuckhardt and Poppe fresh dissection technique to include heart and major vessels. Fetal kidneys were examined and graded for renal papillae development by a method described by Woo and Hoar. The heads from all of the fetuses were examined by a mid-coronal slide. Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described in Dawson. External, visceral and skeletal findings were recorded as developmental variations or malformations.

- 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:

- Chi-square test with Yates' correction factor
- Fisher's Exact test
- Mann-Whitney U-test
- ANOVA (two-tialed) with Dunnett’s test
- Kruskal-Wallis test

Intrauterine parameters (postimplantation loss, live litter size, fetal body weights, fetal sex ratios and numbers of corpora lutea and implantation sites) were assessed as appropriate, on a group mean basis or as a proportional ( % ) litter comparison.

Indices:

Group mean litter basis: postimplantation loss / litter = numer of dead fetuses, resorptions/group per number of gravid females/group

Proportional litter basis: summation per group (%) = postimplantation loss/litter (%)* per number of litters/group
* = number of dead fetuses, resorptions/litter x 100 per number of implantation sites/litter

Summation per group (%) = Viable fetuses affected/litter (%)** per number of litters/group
** = number of viable fetuses affected/litter per number of viable fetuses/litter

Historical control data:

Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased defecation was noted in all dose groups, including the control group. However, the greatly increased frequency of this finding during the dosing period (gestation days 7-19) in the 50 mg/kg/day group was indicative of a treatment-related effect.
Another excreta related finding (feces small in size) was slightly increased in the 50 mg/kg/day group and also appeared to be related to compound administration. This finding occurred at a much lower incidence than the decreased defecation.
Other clinical signs in the treated groups occurred similarly or at a greater frequency in the control group, occurred primarily during the post treatment period or occurred infrequently in single animals. The isolated occurrences of these findings were not suggestive of a treatment-related effect.
[cp. tables 2 and 3 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group female died on gestation day 22. All other animals survived to the scheduled necropsy.
[cp. table 1 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Substantial mean body weight losses occurred in the 50 mg/kg/day group throughout the treatment period (gestation days 7-10, 10-13, 13-19 and 7-19). The differences between the control group and the 50 mg/kg/day were statistically significant at p< 0.01. During the post-treatment period gestation days 19-29, an increased mean body weight gain in the 50 mg/kg/day group was statistically significant at p <0.01 when compared to the control group value. Mean body weights in the 50 mg/kg/day group were slightly lower than the control group values on gestation days 10, 13, 19, 24 and 29. The gestation day 19 value was statistically sigignificant (p< 0.01). Mean gravid uterine weight in the 50 mg/kg/day group was comparable to the control group value. However, the mean net body weight in this group was decreased and the mean net body weight loss was greater than the loss observed in the control group. The difference in net body weight losses was statistically significant at p< 0.05. No adverse effects on mean body weights, body weight changes, gravid uterine weights, net body weights or net body weight losses were observed at dose levels of 2 and 10 mg/kg/day. None of the differences from the control group were statistically significant.
[cp. tables 4, 5 and 6 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption, evaluated as g/animal/day and g/kg/day, in the 50 mg/kg/day group was reduced and statistically significant at p<0.01 throughout the treatment period (gestation days 7-10, 10-13, 13-19 and 7-19) when compared to the control group. Food consumption in this group was comparable to that in the control group during the overall post-treatment period (gestation days 19-29). No adverse effects on food consumption (g/animal/day and g/kg/day) were apparent in the 2 and 10 mg/kg/day groups.
[cp. tables 7 and 8 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean liver weight in the 50 mg/kg/day group was increased 10% when compared to the control group value. The difference was not statistically significant. Mean liver weights in the 2 and 10 mg/kg/day groups were comparable to the control group value. No remarkable differences were observed in mean kidney and spleen weights between the control and treated groups.
[cp. table 9 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the scheduled necropsy, no internal findings related to compound administration were observd at any dose level.
[cp. table 25 in WI-92-154_Individual Data in block "Overall remarks, attachments"]

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic tissue changes in the liver at a dose level of 50 mg/kg/day were comparable to the changes observed in the control group. These changes consisted of cytoplasmic vacuolation and fatty change.
[cp. table 10 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Two females in the 10 mg/kg/day group aborted, one each on gestation days 26 and 27. One 2 mg/kg/day group female aborted on gestation day 29. No abortions were observed at the higher dose level of 50 mg/kg/day and one control group female aborted on gestation day 28. Therefore, no relationship to treatment was apparent.
[cp. table 1 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Postimplantation loss in the 50 mg/kg/day group (1.4 losses/dam) was increased when compared to the control group value (0.5 losses/dam); the difference was not statistically significant. Although the 50 mg/kg/day group value was within the range of the WIL historical control data, only 5 of 61 individual data sets in the historical control data set had mean values equal to or greater than the high dose group value.

A slightly increased (p<0.05) mean number of corpora lutea was observed in the 10 mg/kg/day group.

[cp. tables 11 and 12 in WI-92-287_Summary Tables, WI-92-287_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Total litter losses by resorption:

not specified

Description (incidence and severity):

[cp. tables 11 and 12 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Early or late resorptions:

not specified

Description (incidence and severity):

[cp. tables 11 and 12 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Details on maternal toxic effects:

There were no treatment-related maternal deaths or abortions. One control female died. Abortions occurred in the control (one) , 2 (one) and 10 mg/kg (two) dose groups, but not in the
highest dose group (50 mg/kg); therefore there was no dose response and the abortions were not considered due to treatment. The predominant clinical observation associated with treatment was decreased defecation at the 50 mg/kg/day dose level which was associated with a significantly decreased food intake during the treatment period. Substantial weight loss was noted during the treatment period in the 50 mg/kg/day group, while this group showed significantly increased weight gains in the post-treatment period. There were no gross necropsy findings associated with treatment. There was a slight but not significant increase in mean liver weights at the 50 mg/kg/day dose level, however histopathologic observations of the liver were similar in both control and high– dose rabbits.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

organ weights and organ / body weight ratios

pre and post implantation loss

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weight in the 50 mg/kg/day group (37.2 g) was slightly lower (not statistictiy significant) than the control group value (42.1 g), but was within the range of the WIL historical control data (34.5 -47.8 g). However, only 5 of 61 individual data sets in the historical control had mean values less than the 50 mg/kg/day group value. Fetal growth and survival were unaffected by compound administration at dose levels of 2 and 10 mg/kg/day.
[cp. tables 11 and 12 in WI-92-287_Summary Tables, WI-92-287_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Although postimplantation loss was increased, the mean number of viable fetuses in the 50 mg/kg/day group (7.4 fetuses/dam) was not decreased but was slightly greater than the control group value (6.9 fetuses/dam). This was due to greater mean numbers of implantation sites and corpora lutea in the 50 mg/kg/day group than in the control group. When evaluated on a proportional basis, the value for viable fetuses in the 50 mg/kg/day group (84.6%) was decreased when compared to the control group value (93.8%).
[cp. tables 11 and 12 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The fetal sex ratio in the 50 mg/kg/day group was comparable to that in the control group.

A fetal sex ratio that was slightly skewed toward females (p < 0.05) occurred in the 10 mg/kg/day group. A similar trend was not observed at the higher dose level of 50 mg/kg/day and no relationship to treatment was evident.
[cp. tables 12 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 125(18), 124(18), 122(17) and 147(20) in the control, 2, 10 and 50 mg/kg/day groups, respectively. The numbers of fetuses (litters) with malformations were 0(0), 3(3), 3(3) and 4(4) in the same dose groups, respectively.

The only external malformation observed in this study was macroglossia in one 10 mg/kg/day group fetus (no. 13945-5). No external developmental variations were observed in fetuses at any dose level.
[cp. tables 13-17 in WI-92-287_Summary Tables and also WI-92-287_Individual Data attached in block "Overall remarks, attachments"]

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were observed in O, 3, 3 and 2 fetuses in the control, 2, 10 and 50 mg/kg/day groups, respectively. In the 50 mg/kg/day group, two fetuses (nos. 13870-8 and 13942-3) had spherical enlargements on right rib nos. 7, 8 and/or 9. The percentage of fetuses affacted in the 50mg/kg/day group (1.4%) was within the range of the WIL historical control data (0.0 % - 1.9%). An extra site of ossification anterior to sternebra no. 1 was observed in one 2 mg/kg/day group fetus (no. 13922-4) and two 10 mg/kg/day group fetuses (nos. 13882-4 and 13932-1). One fetus in each of the 2 and 10 mg/kg/day groups had vertebral anomalies without associated rib anomalies. For fetus no. 13885-9 in the 2 mg/kg/day group, these anomalies consisted of extra lumbar arches and centra and lumbar arches and centra that were located more anterior or more posterior than normal. For fetus no.13945-5 in the 10 mg/kg/day group, these anomalies consisted of thoracic centra that were absent, smaller than normal or located more anterior or more posterior than normal. The remaining skeletal malformation was observed in 2 mg/kg/day group fetus no. 13905-7. This fetus had rib anomalies, including a malformed rib, a bifurcated rib and fused costal cartilage. The percentages of fetuses with individual skeletal malformations in the 2 and 10 mg/kg/day groups were within the ranges of the WIL historical control data. Skeletal variants occurred in all dose groups, including the control group, and consisted primary of 13th full and rudimentary ribs and 27 presacral vertebrae. No remarkable differences were observed between the control and treated groups in the percentages of fetuses (litters) affected. Other skeletal variants in the treated groups occurred at a similar frequency in the control group, were observed at a limited frequency (in one or two fetuses per group),and/or were within the ranges of the WIL historical control data. No relationship to treatment was evident.
[cp. tables 13-17 in WI-92-287_Summary Tables, WI-92-287_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissues malformations were observed only in the 50 mg/kg/day group. Cataracts were noted in both eyes of fetus no. 13866-5. Heart and great vessel anomalies were observed in another 50 mg/kg/day group fetus (no.13901-2). These anomalies consisted of a bulbous ascending aorta and aortic arch, a rudimentary pulmonary trunk, md an interventricular septal defect. The percentages of fetuses (litters) with these malformations in the 50mg/kg/day group were within the ranges of the WIL historical control data. The primary soft tissue development variations in all groups, including the control group, consisted of non dose-related occurrences of accessory spleens and major blood vessel variations (in all cases the Ieft carotid arose from the brachiosephalic trunk). Other soft tissue variants in the treated groups (a hemorrhagic ring around the iris, retrocaval ureters, renal papillae not developed and a small gallbladder) occurred infrequently, occurred similarly in the control group and/or within the ranges of the WIL historical control data. No relationship to treatment was evident.
[cp. tables 13-17 in WI-92-287_Summary Tables, WI-92-287_Individual Data and Historical Control Data attached in block "Overall remarks, attachments"]

Details on embryotoxic / teratogenic effects:

Postimplantation loss (%) was increased and the mean fetal weight was decreased at the 50 mg/kg/day dose level but the differences from control were not statistically significant. There were no adverse effects on intrauterine growth or survival at the 2 or 10 mg/kg/day dose levels. Although there were a few malformations seen in the treated groups which were not present in the concurrent control, there was no dose relationship in the types of malformations which would indicate a teratogenic effect of the test material. The types of malformations seen were within the historical range of the performing laboratory.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

SUMMARY OF EXTERNAL. VISCERAL AND SKELETAL EXAMINATIONS

No indication of developmental toxicity was expressed at dose levels of 2, 10 and 50 mg/kg/day upon evaluation of the fetal morphological data. Malformations were observed in 0, 3, 3 and 4 fetuses in the control, 2, 10 and 50 mg/kg/day groups, respectively. However, no dose-relationship was apparent in the types of malformations present in the treated groups. The variations expressed in the treated groups were not present in a dose-dependent manner but were generally similar to those present in the control group. 

Applicant's summary and conclusion

Executive summary:

This study was conducted to obtain an assessment of the potential of the substance to induce maternal and embryo/fetal toxicity in rabbits. The guideline requirements of EPA OPP §83-3, 1984 (eq. to OECD TG 414, 1981) were fulfilled. The test material was suspended in 0.5% aqueous methylcellulose and administered daily by gavage (5 ml/kg) to 4 groups of 20 artificially inseminated New Zealand White rabbits during gestation days 7 through 19. Dose levels were 0, 2, 10 and 50 mg/kg/day. No adjustment for purity was made in preparation of the dosing suspension. All dosing suspensions were prepared twice during the study and stored at room temperature until used. Analysis of the dosing suspensions confirmed that all group suspensions contained the targeted concentration. The homogeneity of test article in dosing suspensions was confirmed prior to administration of the first dose and stability was verified by analyzing the lowest and highest concentrations on the last day of dosing. Food and water were available ad libitum. The animals were observed at least twice daily for mortality and gross signs of toxicity. Detailed clinical observations were recorded daily. Body weights were recorded on gestation days 0, 7, 10, 13, 19, 24, and 29 and food consumption measured during days 0-29. Gross necropsies were conducted on all animals that died during the study or that were sacrificed after aborting. All surviving animals were sacrificed on gestation day 29. Gross postmortem examinations were conducted and liver, kidney, spleen and uterus weights were determined. Livers from control and high-dose does were examined histopathologically. The uterus and ovaries were examined, and the number and location of viable and nonviable fetuses, early and late resorption, total implantations and corpora lutes were recorded. Fetuses were weighed and examined for external malformations and variations. Crown-rump lengths were recorded for late resorption and then the tissues were discarded. Each viable fetus was examined viscerally to determine the sex and to assess development of internal organs including heart, kidney and brain. Skeletal development was evaluated following staining with Alizarin Red S. There were no treatment-related maternal deaths or abortions. One control female died. Abortions occurred in the control (one), 2 (one) and 10 mg/kg (two) dose groups, but not in the highest dose group (50 mg/kg); therefore there was no dose response and the abortions were not considered due to treatment. The predominant clinical observation associated with treatment was decreased defecation at the 50 mg/kg/day dose level which was associated with a significantly decreased food intake during the treatment period. Substantial weight loss was noted during the treatment period in the 50 mg/kg/day group, while this group showed significantly increased weight gains in the post-treatment period. There were no gross necropsy findings associated with treatment. There was a slight but not significant increase in mean liver weights at the 50 mg/kg/day dose level, however histopathologic observations of the liver were similar in both control and high–dose rabbits. Postimplantation loss (%) was increased and the mean fetal weight was decreased at the 50 mg/kg/day dose level but the differences from control were not statistically significant. There were no adverse effects on intrauterine growth or survival at the 2 or 10 mg/kg/day dose levels. Although there were a few malformations seen in the treated groups which were not present in the concurrent control, there was no dose relationship in the types of malformations which would indicate a teratogenic effect of the test material. The types of malformations seen were within the historical range of the performing laboratory. In conclusion, the substance produced maternal toxicity at the highest dose level tested, 50 mg/kg/day, as indicated by weight loss and decreased food consumption. Slight developmental toxicity, as indicated by slightly increased postimplantation loss and slightly decreased fetal weights, was seen in the 50 mg/kg/day dose group. There were no developmental malformations associated with exposure to the substance in this study. The No Observable Effect Level (NOEL) for maternal and developmental toxicity was therefore 10 mg/kg/day.