Reaction mass of 3-({[2,2-dimethyl-3-({[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethoxy]carbonyl}oxy)propoxy]carbonyl}oxy)prop-1-ene and 3-{[(2,2-dimethyl-3-{[(prop-2-en-1-yloxy)carbonyl]oxy}propoxy)carbonyl]oxy}prop-1-ene and diallyl 2,2'-oxydiethyl dicarbonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-01-08 to 1985-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl 2,2'-oxydiethyl dicarbonate

EC Number:

205-528-7

EC Name:

Diallyl 2,2'-oxydiethyl dicarbonate

Cas Number:

142-22-3

Molecular formula:

C12H18O7

IUPAC Name:

diallyl 2,2'-oxydiethyl dicarbonate

Details on test material:

- Substance type: pure active substance
- Physical state: clear colorless liquid
- Stability under test conditions: known to be stable if not heated or exposed to moisture.
- Storage condition of test material: at room temperature out of direct light.
- Other: specific density 1.143.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Dutchland Inc., Swampbridge Road, Denver, PA.
- Age at study initiation: 22-23 weeks
- Weight at study initiation: ranged from 3.2 to 4.3 kg
- Fasting period before study: no
- Housing: individually in mesh-bottomed stainless steel cages
- Diet (e.g. ad libitum): ad libitum (free access to pelleted chow (Purina Certified Rabbit Chow No. 5332, batches No. NOV 15842C, NOV 20841A, JAN 06842A, JAN 19841A, JAN 19842A).
- Water (e.g. ad libitum): ad libitum (free access to fresh municipal tap water)
- Acclimation period: 4 weeks prior to insemination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 +- 3°C
- Humidity (%): 50 +- 20% (an occasional fluctuation of the relative humidity above 70% was not considered to have affected the outcome of the study)
- Air changes (per hr): data not available
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To: data not available

Administration / exposure

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: not diluted. The doses administered was based on the most recent body weight of each rabbit.

PREPARATION FOR DOSAGE:
All animals were shaved on approximately day 0 of gestation. Hair was removed from an area of the dorsum at least 4 inches wide and 9 inches long.

METHOD OF DOSAGE
Groups of 18 inseminated rabbits were treated, by dermal application, with the test substance at dosages of 0.1, 0.5 or 1.0 mL/kg/day, for 6 hours per day, from day 6 to day 18 of gestation, inclusive. A control group was treated in an identical manner except that it was dosed with sterile isotonic saline at a volume of 1.0 mL/kg/day. The test liquid or control solution was spread on the shaved area of the back. Restraint collars were fitted on each animal immediately prior to dosage. After 6 hours, any residual compound or control solution was removed by blotting the dosage area with nonabsorbent cotton and the restraint collars were removed.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 114, 572, and 1143 mg/kg/day
- Concentration (if solution): at respective dose volumes of 0.1, 0.5, and 1.0 mL/kg.
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 grams/mL.
DIET PREPARATION Not applicable
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


CONTROL MATERIAL
The control article, sterile isotonic saline (0.9% sodiurn chloride for injection U.S.P.), supplied by Abbott Laboratories

- Amount: at the same dosage volume as treated animals in the high dose group (1 mL/kg bw/day)
- Lot/batch no. (if required): 54-542-DM-08 and 59-430-NA-3.
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Five-gram samples of the test substance were retained and subsequently shipped to the sponsor for analysis to confirm identity at the start of treatment, during the treatment period and at the end of the study. Viscosity measurements of the sample returned at the end of the study were within the normal specification.

Details on mating procedure:

- Impregnation procedure: [artificial insemination]
- If cohoused: not applicable
Female rabbits were luteinized with an intravenous injection of 50 IU of chorionic gonadotropin 19 days prior to insemination. Two to four hours prior to insemination, a second 50-1U dose of chorionic gonadotropin was administered to each female. Sperm samples were collected from stud bucks, and a diluted pooled sample was prepared. Each day samples from at least 4 males were used; in all a total of 18 males were utilized during the study. Each female was inseminated with at least 0.6 mL of the pooled sample, which contained in excess of 20 Mio spermatozoa/mL. Insemination was performed on January 2, 3, 7, 8, 9 and 10, 1985
- Verification of same strain and source of both sexes: data not available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: data not available
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

from day 6 to day 18 of gestation, inclusive.

Frequency of treatment:

each day

Duration of test:

6 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.1 mL/kg bw/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.5 mL/kg bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.0 mL/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

18 female animals

Control animals:

other: 1ml/kg bw/day of 0.9% sodium chloride for injection U.S.P.

Details on study design:

- Dose selection rationale: Dosages were selected based upon the data from a pilot study (Range-Finding Teratology Study by Dermal Application in the Rabbit, Bio-Research Project No. 81981). In this study groups of 5 inseminated rabbits were treated by dermal application with the test substance at dosages of 0.1, 0.5, 1.0 or 3,0 mL/kg/day 24 hours per day from day 6 to day 18 of gestation, inclusive. The dosage site was "occluded" with gauze and the animals were fitted with a restraining collar. Maternal toxicity was evidenced in all test substance-treated groups by skin reactions at the treatment site characterized by dark red/black areas which subsequently showed scab formation. Systemic toxicity was shown at the 3.0 mL/kg/day dose level where 1 rabbit was killed in poor condition and the remaining rabbits had significant weight loss following completion of dosage (day 18 to day 24 of gestation). Body weight losses were seen in all groups (treated and control) during the dosage period which suggest that these resulted from the dosage procedure (24-hour occlusion with restraining collars). Similarly, generally elevated abortion rates and increased incidences of late resorptions may have been related to this dosage procedure. Fetal malformations (hydrocephaly and abnormal limb/paw flexure) occurred among fetuses in the 0.1, 0.5 and 1.0 mL/kg/day groups (there were no live litters in the 3.0 mL/kg/day group).
- Rationale for animal assignment (if not random): randomized

Examinations

Maternal examinations:

All animals were checked for mortality or abnormal condition each day for any adverse reactions to treatment, indications of poor health and abnormal behavior from day 6 of gestation until day 18 of gestation. These examinations were performed prior to dosage and at least once post-dosing. In addition, the skin at the treatment site was examined daily. Any signs of abortion of premature delivery were recorded. Does which aborted or littered early were sacrificed. All aborted material was examined. All fetuses aborted prior to day 27 of gestation were examined externally and preserved in neutral buffered 10% formalin. All fetuses aborted on and after day 27 of gestation or littered early were examined as for fetuses at termination.


CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Throughout the acclimation and gestation period
- Cage side observations checked in table [No.2] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the acclimation and gestation period


BODY WEIGHT: Yes
- Time schedule for examinations: In addition to the weight assessment on arrival and weekly during the acclimation period, female rabbits were weighed on days 0, 6, 9, 12, 15, 18, 24 and 29 of gestation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data Not applicable
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

Food consumption and fecal volume are qualitative assessed
-Calculation: not noted

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data. Not applicable
- Time schedule for examinations:


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
Any animal which was sacrificed during the study was killed and was then given a complete gross pathological examination. The uterus was removed and examined. Any live or dead fetuses were examined externally and were preserved in neutral buffered formalin for does sacrificed prior to day 27 of gestation. For does examined on or after day 27 of gestation, live and dead fetuses were examined as for fetuses at cesarean section. Abnormal tissues were retained in neutral buffered 10% formalin.
OTHER:
Samples of tissue from the treatment site, two liver lobes and any abnormal tissues were preserved for further examinations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
The uterus was removed, and the number and position of live fetuses, dead fetuses, empty implantation sites and early, middle and late resorptions were recorded. (Early resorption: metrial gland with some piacental/fetal tissue present. Middle resorption: both fetal and placental tissue present. Late resorption: recognizable fetal structure present. Empty implantation site: implantation site scar present, but no fetal or placental tissue.)

Fetal examinations:

On day 29 of gestation the fetuses were removed from the uterus and weighed, and were then killed
- External examinations: Yes: [all per litter] and also any live or dead fetuses for does sacrificed prior to day 27 of gestation. For does examined on or after day 27 of gestation, live and dead fetuses were examined as for fetuses at cesarean section.
- Soft tissue examinations: Yes. Samples of tissue from two lobes of the liver from 1 male and 1 female fetus per litter were preserved in neutral buffered 10% formalin.
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes: [1/3 per litter ]
Other:
- Body weight: Yes [all per litter]

Statistics:

Statistical analyses were performed upon both the body weights for all does and the body weights from only those pregnant with live litters at term. The group mean (S.D.) body weights and weight gains were calculated, and statistical analysis was performed using a one-way analysis of variance. Where the F value was found to be significant P <0.05), differences between control and treated groups were analyzed using Student's 't' test.

Indices:

MATERNAL DATA: Pregnancy and abortion rates were calculated
UTERINE FINDINGS AND FETAL DATA: Pre- implantation loss (%) and post-implantation loss (%)

Historical control data:

Historical control data (Rabbit NZW Dutchland 1983-1984 No. 81982) were provided in the key study and contain the same investigated endpoints as in the present study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment of pregnant rabbits with the test substance at dosages of 0.1, 0.5 or 1.0 mL/kg/day by unoccluded dermal application resulted in decreased food intake, weight loss, abortion at 0.5 and 1.0 mL and mortality at the 1.0 mL/kg/day level. Necropsy findings among the 1.0 and 0.5 mL/kg bw/day treated group included a variety of adversed effects in organs as well as significant high abortion rates.

Effect levels (maternal animals)

Dose descriptor:

LOEC

Effect level:

0.1 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: only at doses with severe maternal toxicity

Details on embryotoxic / teratogenic effects:
Fetal findings in the 0.5 and 1.0 mL/kg/day groups of ocular opacities, small lens(es) and associated anomalies are indicative of embryotoxic effects during the later stages of ocular development. Also in the 0.5 mL/kg/day group incidental sceletal abnormalities were detected.

Effect levels (fetuses)

Dose descriptor:

NOEC

Effect level:

0.1 mg/kg bw/day (nominal)

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Mortality

In the 1.0 mL/kg/day treated group, 6 rabbits died and 1 was killed in a moribund condition.

Three of these rabbits (Nos. 457, 461 and 468) died after 1 or 2 days of treatment. Clinical observations for rabbit 461 included not eating and a red wet area of scab formation at the dosage site; there were no clinical findings for rabbits 457 and 468. Gross pathological findings common to all 3 rabbits were dark area(s) on the mucosa of the stomach, red discoloration or clot in the vaginal serosa and dilatation or red discoloration of the urinary bladder. For rabbit 468 pale areas were noted on the endocardium and pale foci on the cortices of the kidneys. Rabbits 453, 463 and 464 died on days 22, 21 and 20 of gestation, respectively. All three rabbits had not been eating at times during the dosage period, and on the 2 days prior to death rabbit 463 had been weak and thin and on the day prior to death rabbit 464 had labored respiration. At necropsy, findings in the stomachs included pale/dark/ depressed area(s) on the mucosa of the stomach for all those rabbits; there was a perforation in the stomach of rabbit 453. The digesta of rabbit 463 was dark and firm. Rabbit 464 had dark frothy fluid in the trachea and uncollapsed lungs which were reddened; in the thoracic cavity of rabbit 453 there was brownish fluid. Both rabbits 463 and 464 had generalized pallor and firmness of the liver with multiple pale foci; in addition, rabbit 463 had a yellowish pale area extending into the parenchyma. Rabbit 463 had multiple dark areas on the kidneys. Rabbit 460 was killed in a moribund condition of day 22 of gestation. Clinical signs on this day included yellow nasal discharge, labored respiration, coldness and prostration. Necropsy observations included dry thickened areas at the dose site, pale foci on the left lobe of the liver, multiple dark depressed areas on the mucosae of the stomach and duodenum and dark firm digesta in the large intestine.

Clinical Findings

Among rabbits in the treated groups, dark red/black discrete or diffuse areas, sometimes preceded by reddening of the skin, were commonly seen at the dosage sites. For some rabbits in all test substance-treated groups, red secretions from the black thickened areas were noted. These lesions often subsequently developed yellow coloration at their periphery and scab formation. The area of the dosage site affected by black thickened lesions was increased in a dose-related fashion (it should be noted that dosage was varied by adjustment of dose volume). Qualitative assessment of food consumption and fecal volume indicated dose-related increased incidences in the 0.5 and 1.0 mL/kg/day dose groups during the treatment period and for the period day 19 to day 24 of gestation. Reddening of the dorsal cervical region and pinna was seen for some rabbits in the 1.0 mL/kg/day group. One doe in the 0.5 mL/kg/day group and 2 does in the 1.0 mL/kg/day group had reddening or swelling in the urogenital area.

Body weights

Rabbits (including all animals in the group) in the 1.0 mL/kg/day group had significant weight losses throughout the dosage period (day 6 to day 18 of gestation), with weight losses continuing between days 18 and 24 of gestation. These weight losses resulted in significantly lower body weights on days 12, 15, 18 and 24 of gestation. Consideration of all rabbits in the 0.5 mL/kg/day group showed weight losses or slightly lower body weight gains than the control group during assessment intervals of the dosage period, resulting in a significant (P<0.01) overall weight loss between days 6 and 18 of gestation. For those rabbits in the 0.5 mL/kg/day group which had live litters on day 29 of gestation, there was an overall weight loss between days 6 and 18 of gestation. The body weights and body weight gains of the 0.1 mL/kg/day treated group were similar to control values.

Gross pathological findings

Two, 0, 3 and 8 rabbits were sacrificed preterminally due to abortion or littering early in the control, 0.1, 0.5 or 1.0 mL/kg/day groups, respectively. A further 6 rabbits died and 1 rabbit was sacrificed in a moribund condition in the 1.0 mL/kg/day group. The remaining rabbits were examined at cesarean section on day 29 of gestation. Pale foci on the liver were noted for 5 rabbits in the 1.0 mL/kg/day treated group dying or sacrificed between days 20 and 22 of gestation. Firmness and/or an irregular surface of the liver occurred for 1 doe in the 0.5 mL/kg/day group and 4 does in the 1.0 mL/kg/day group which died or were sacrificed. Four rabbits in the 1.0 mL/kg/day group dying or sacrificed preterminally had pale foci or pale area(s) on the heart. Three of these 4 rabbits and 1 further rabbit in this group had pale foci or pale areas on the kidneys. Pale foci or pale areas on the mesentery occurred for 1 doe in the 0.5 mL/kg/day group and 4 does in the 1.0 mL/kg/day group. In all test substance-treated groups skin lesions including areas of the dosage site which were dark, pale, depressed, dry, thickened and/or with scab formation were noted at necropsy. Some animals in all groups had findings in the stomach which included dark areas/foci, depressed areas (ulceration) and/or thickening. There was a slightly higher incidence of these findings in the 1.0 mL/kg/day group which may be correlated with the greater occurrence of rabbits not eating in this group. (It is also worthy of note that the majority of animals in the 1.0 mL/kg/day dose group with stomach lesions had been found dead.) Dark ingesta/digesta, probably indicative of bleeding from areas of ulceration, was noted for 4 rabbits in the 1.0 mL/kg/day group, and 1 other rabbit had a perforation of the stomach. The incidence of prominent lobular architecture and pallor in the control and treated groups indicated these findings were incidental. The remaining gross pathological findings were judged to be incidental or agonal.

Abortion

One, 0, 3 and 6 does in the control, 0.1, 0.5 and 1.0 mL/kg/day dose group, respectively, aborted. In addition, 1 doe in the control group and 2 does in the 1.0 mL/kg/day treated group littered early.

Details on maternal toxic effects (Uterine findings)

The pregnancy rate was at least 88.9% in all groups. There were 3 females in the 1.0 mL/kg/day dose group examined on day 29 of gestation, the remainder having died or been sacrificed due to abortion, littering early or poor condition. The ovarian and uterine parameters including numbers of corpora lutea, implantation sites, live fetuses, dead fetuses and resorptions, the fetal and litter weights, uterine weights and the pre- and post-impl antation losses in the 0.1 and 0.5 mL/kg/day treated groups were similar to control values for those does alive on day 29 of gestation. Two of the 3 does which aborted in the 0.5 mL/kg/day group had a number of resorptions in the uterus. For those does which aborted, littered early, died or were sacrificed preterminally in the 1.0 mL/kg/day treated groups, resorptions were frequently observed either as aborted material or in utero.

Fetal findings

a) Major Malformations

The incidence of major malformations in the treated groups, among 1itters from females examined at cesarean section, was not significantly different from control values.

b) Minor Visceral Anomalies

A significantly lower overall incidence of fetuses with minor visceral anomalies in the 0.1 mL/kg/day dosed group was considered to be unrelated to dosage. There was a significant (P<0.001) increase in the incidence of ocular opacities and small lenses in the 1.0 mL/kg/day treated group 5 fetuses from 1 of the 3 litters examined at term being affected. Three of these fetuses also had lens(es) formed in 2 layers. A significant (P<0.01) number of fetuses (6 fetuses from 3 litters) in the 0.5 ml/kg/day group had small lens(es). Some of these fetuses had opacities of the lens (an external observation), lens(es) formed in 2 layers, encapsulated with or connected to retinoid/choroid/corneal tissue. c) Minor Skeletal Anomalies

The overall incidences of litters and fetuses affected in the treated groups were unaffected. Significantly (P<0.05) elevated incidences of fetuses with absent pubic bones and reduced numbers of phalanges and/or metatarsals in the hindpaws in the 0.1 mL/kg/day treated group were not attributed to dosage as all the affected fetuses were from 1 litter.

d) Common Skeletal Variants

The incidence of sternebral variants in the treated groups was not significantly different from control values. There was a significant (P< 0.05) decrease in the occurrence of single thirteenth ribs and a concomitant increase in the incidence of paired thirteenth ribs in the 0.5 mL/kg/day along with a significant (P<0.05) elevation in the incidence of 27 presacral vertebrae.

e) Fetuses from Aborted Litters

Among abortuses recovered on days 19 to 22 of gestation from the 1.0 mL/kg/day group, open eye(s), domed skull and eventration of the intestines and liver at the umbilicus were common findings. Two abortuses (454/1 and 3) had clefts in the vertebral column. Since the eyelids are normally closed by day 20 of gestation, this finding in fetuses aborted and found dead, which may have been dead in utero, may be indicative only of their stage of development. Similarly the findings of domed skull (fetuses were examined on criteria for fetuses on day 29 of gestation) could be reflective of their gestational age. In considering the remaining observations, the possibility that these findings may have resulted from traumatic injury precludes a definitive assessment of their relationship to test substance treatment.

Applicant's summary and conclusion

Conclusions:

Treatment of pregnant rabbits with the test substance at dosages of 0.1, 0.5 or 1.0 mL/kg/day by unoccluded dermal application resulted in decreased food intake, weight loss, abortion and mortality at the 1,0 mL/kg/day level.
Necropsy findings among the 1.0 mL/kg/day treated group animals included pale foci, firmness and/or irregular surface of the liver and, for the heart, kidneys and mesentery, pale foci and/or pale area(s). Maternal toxicity at the 0.5 mL/kg/day level was evidenced by lower food consumption and overall weight loss during the treatment periods (day 6 to day 18 of gestation). Also in the 0.5 mL/kg/day group 3 rabbits aborted, and gross pathological findings in the liver (firmness/irregular surface) and mesentery (pale foci) each occurred once. Some rabbits in all test substance-treated groups had skin lesions, reddening, dark red/black areas and scab formation. Uterine findings for pregnant does surviving to day 29 of gestation, including 3 rabbits in the 1.0 mL/kg/day, were unaffected.
However, among rabbits in the 1.0 mL/kg/day group which aborted, died or were sacrificed in a moribund condition, there was a high incidence of resorptions indicative of embryolethality. Fetal findings in the 0.5 and 1.0 mL/kg/day groups of ocular opacities, small lens(es) and associated anomalies are indicative of embryotoxic effects during the later stages of ocular development. Also in the 0.5 mL/kg/day group the incidence of single thirteenth ribs was decreased and the occurrence of paired thirteenth ribs was slightly increased, combined with an increased incidence of 27 presacral vertebrae. These skeletal findings, when accompanying adverse effects upon maternal weight gain, are not considered to be of teratological significance. Among abortuses in the 1.0 mL/kg/day group, eventration of the intestines and liver were common observations. While these may be related to treatment, there is the possibility that they were related to trauma associated with the abortion process. There was no evidence of embryolethality or embryotoxicity at the 0.1 mL/kg/day dose level.

Executive summary:

Diallyl 2,2'oxydiethyl dicarbonate, was administered by dermal application to groups of gravid rabbits throughout major organogenesis to assess its teratogenic potential.

Groups of 18 inseminated rabbits were treated, by dermal application, with the test substance at dosages of 0.1, 0.5 or 1.0 mL/kg/day, for 6 hours per day, from day 6 to day 18 of gestation, inclusive. A control group was treated in an identical manner except that it was dosed with sterile isotonic saline at a volume of 1.0 mL/kg/day. Six rabbits died and 1 was killed in a moribund condition in the 1.0 mL/kg/day treated group. No other animals died or were sacrificed in a moribund condition. There were skin lesions at the dosage sites for some rabbits in all test substance-treated groups which were typified by dark red/black thickened areas which subsequently turned yellow and showed scab formation. The area affected was related to dosage. The incidence of rabbits not eating and/or having decreased fecal volume (quantitatively assessed) was increased in the 0.5 and 1.0 mL/kg/day group. The 1.0 mL/kg/day group had significant weight losses during all intervals in the treatment period (day 6 to day 18 of gestation) and between days 18 and 24 of gestation. In consequence the body weights were significantly less than those of the control group on days 12, 15, 18 and 24 of gestation. There was a marked weight loss between days 6 and 18 of gestation in the 0.5 mL/kg/day group. The growth of the 0.1 mL/kg/day group was similar to that of the controls. Pale foci, firmness and/or an irregular surface were common findings for the liver of animals in the 1.0 mL/kg/day group. Other findings seen at necropsy for this group (1.0 mL/kg/day) included pale foci or pale area(s) on the heart, kidneys and/or mesentery of some rabbits. In the 0.5 mL/kg/day group 1 rabbit had firmness and an irregular surface to the liver and a second rabbit had pale foci on the mesentery. One, 0, 3 and 6 does in the control, 0.1, 0.5 and 1.0 mL/kg/day treated groups, respectively, aborted. The pregnancy rate in all groups was at least 88.9%. The ovarian and uterine parameters (number of corpora lutea, implantation sites, live fetuses, dead fetuses, resorptions, fetal weights and pre- and post- implantation losses) of the 0.1 and 0.5 mL/kg/day treated does were unaffected. For the 1.0 mL/kg/day group there was a high level of resorptions observed for animal s dying, aborting, Uttering early or sacrificed preterminally, but the uterine parameters of the 3 females in this group alive on day 29 of gestation were similar to control values.

In the 0.5 and 1.0 mL/kg/day groups significant (P <0.01 and P <0.001, respectively) numbers of fetuses had small lens(es), In the 0.5 mL/kg/day group 6 -fetuses from 3 litters were affected and in the 1.0 mL/kg/day group 5 fetuses from 1 litter were affected. All the affected fetuses in the 1.0 mL/kg/day group and some in the 0.5 mL/kg/day group also had ocular opacities. Also among these fetuses there were other ocular findings. The overall incidence of minor visceral anomalies was not significantly different from control values. The incidences of major malformations and minor skeletal anomalies among term fetuses were not significantly different from control values. Findings of eventration of the liver and intestines among abortuses from 3 litters in the 1.0 mL/kg/day group were possibly related to trauma in the abortion process. The incidence of major malformations in the treated groups, among 1itters from females examined at cesarean section, was not significantly different from control values. In the 0.5 mL/kg/day group there was a significant (P <0.05) decrease in the incidence of single thirteenth ribs and an accompanying increase in the incidence of paired thirteenth ribs and a significant increase in the incidence of 27 presacral vertebrae. The incidence of sternebral variants in the treated groups was not significantly different from control values.

The test substance was found to produce maternal toxicity in terms of skin lesions at the treatment site at levels of 0.1, 0.5 and 1.0 mL/kg/day. Systemic toxicity occurred at the 0.5 and 1.0 mL/kg/day group, with the majority of rabbits in the 1.0 mL/kg/day group either dying or aborting. Maternal toxicity at the 0.5 and 1.0 mL/kg bw evidenced by body weight loss. Embryotoxicity was evidenced by significantly increased ocular anomalies in the 0.5 and 1.0 mL/kg/day groups. These findings when accompanying significant adverse effects upon maternal weight loss, are not considered to be of teratological significanceThere was some indication of embyrolethality among those rabbits in the 1.0 mL/kg/day group which died or aborted. Neither embryolethality nor embroyotoxicity occurred at the 0.1 mL/kg/day dose level.