"Husbandry:

Housing: Individually, except during mating, in stainless steel suspended cages with wire mesh floors, tops and fronts and sub-cage floor pans. During exposure intervals, animals were individually housed in stainless steel wire mesh partitioned areas within a wahmann 1m3 glass and stainless-steel exposure chamber. Placement of each animal within the partitioned areas of the chambers was rotated daily to assure uniform exposure of the animals.

Food:

Feed was withheld on the day of arrival. On the first, second and third full days in-house the animals received 25, 50 and 100 grams of Purina Certified Rabbit Chow No. 5325 (High-Fiber) feed, respectively; thereafter, 150 grams of the food was fed per day during the acclimation period. During gestation, animals were fed ad libitum; however, during daily exposure intervals, animals did not have access to food.

Water:

Ad libitum; tap water (Elizabethtown Water Company) to individual cages by an automated water delivery system during the non-exposure period. During exposure intervals, animals did not have access to water.

There were no known contaminants present in the food or water capable of interfering with the results of this study.

Environmental Conditions:

The following presentation pertains to the animal quarters; see Appendix M (Inhalation Report) for environmental conditions recorded in the chambers during the exposure intervals.

Photoperiod:

12 hour light/dark cycle (light cycle was approximately 7 AM to 7 PM) via automatic timer.

Temperature:

Recorded twice daily (morning and afternoon).

Desired: 60-70°F (16-21°C)

Actual: 58-74°F (14-23°C)

Relative Humidity:

Recorded once daily (morning).

Desired: 40-60%

Actual: 16-63%

Identification:

Each mated female rabbit sorted into test groups was identified with a metal ear tag bearing its animal number. This individual animal number plus Bio/dynamics' project number comprised a unique identification number.

Assignment to Groups:

A sufficient number of female rabbits was received so as to discard, if necessary, unhealthy females prior to initiation of mating. The population of animals received for study was evaluated for suitability to use on test by the staff veterinarian prior to the initiation of mating. Females which mated were assigned to the groups daily in such a way as to provide an equal distribution of mated females among groups and equalize, as best possible, the Day 0 gestation mean group body weights.

Mating:

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 second, different male. When coitus was observed with the second male, the female was considered mated and returned to her cage. The day on which evidence of mating was observed with both males was defined as Day 0 of gestation.

Mating was scheduled to provide Day 30 gestation sacrifices during the Monday to Friday work week. The number of females mated daily was also controlled to limit the number of mated females available for sorting into groups on a particular day. In this study, the maximum number of mated females sorted into groups on a particular day was 10.

Test Substance Administration:

Route:

Whole body inhalation.

Justification for Route of Administration:

The route of administration of the test substance was determined by the sponsor and represents one of the potential routes of human exposure.

Chamber Operation Exposure Procedures, Atmospheric Sampling and Analytical Data:

See Inhalation Report (Appendix M).

Treatment Schedule:

Animals were exposed for 6 hours/day on Days 6-18 of gestation. Animals remained in the chamber for a minimum of 30 minutes following each exposure session to permit the test gas to be cleared prior to removing the animals.

Dates of Exposure:

Day 6 of gestation:

4, 5 and 6 January 1991.

Day 18 of gestation:

16, 17 and 18 January 1991

In-Life:

Physical Observations:

(Methodology and References, Appendix A). Twice daily (morning and afternoon) for signs of pharmacologic or toxicologic effects and mortality. Physical observations for abnormal signs were performed once during each exposure for all animals as a group starting on the second exposure day. In addition, each female was given a detailed physical examination or. Days 0, 6-18, 24 and 30 of gestation. On Days 6-18 of gestation, the detailed physical examinations were conducted after all animals had been removed from the chamber following exposures.

Body Weights:

(Methodology and References, Appendix A)

Recorded on Days 0, 6, 9, 12, 15, 18, 24, and 30 of gestation. Day 30 gestation body weights are presented as actual and corrected (the actual Day 30 gestation body weight minus the weight of the gravid uterus).

Food Consumption:

(Methodology and References, Appendix A)

Recorded for the following intervals during gestation: 0-1, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 13-14, 16-17, 18-19, 19-20, 24-25 and 29-30.

Postmortem:

Maternal Evaluations:

Gross Postmortem Examinations:

Complete gross postmortem examinations were performed on all study rabbits. External surface, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs were examined for all animals.

The carcass of each female was discarded at completion of the gross postmortem evaluation.

Organs Weighed:

(Methodology and References, Appendix A)

The liver was weighed for all females at gestation Day 30 sacrifice, and both absolute and relative weight data are presented. The latter was presented relative to the corrected Day 30 gestation weights.

Animals Killed:

Day 30 of gestation.

Method:

Intravenous injection of Sodium Pentobarbital via the marginal ear vein.

Dates:

28 through 30 January 1991.

Reproductive System:

The intact uterus (ovaries attached) was removed from the abdominal cavity, weighed and the number and location of the following were recorded for each uterine horn; live fetuses; dead fetuses (no evidence of tissue degeneration); late resorptions (recognizable dead fetus undergoing degeneration regardless of size); early resorptions (evidence of implantation but no recognizable fetus:); implantation sites; The ovaries were dissected free from the uterus and evaluated fur the presence and number of corpora lutea. When no uterine implants were grossly apparent, the uterus was stained with ammonium sulfide (method of Salewski see Appendix A. page A-2). When no foci were visualized post-staining, the female was considered not pregnant.

Fetal Evaluations:

All fetuses were given a gross examination for external malformations variations to include observation for palatal defects. Subsequently, each fetus was weighed, humanely sacrificed via an intraperitoneal injection of pentobarbital sodium and discarded.

Late Resorptions:

Late resorptions were weighed, examined grossly for external malformations and discarded.

Statistical Analysis:

Due to the small group sizes involved, data were not evaluated statistically.

Protocol Deviations:

The following protocol deviations were not considered to have adversely effected animal health and were not considered to have compromised the validity or integrity of the study. Temperature and relative humidity values within the animal quarters and exposure chambers were outside of the desired range on some occasions."

P0 Female New Zealand Rabbits