"Species/Strain and Reason for Selection:

The Crl:CD®(SD)IGS BR VAF/Plus® rat was selected as the Test System because:1) it is one mammalian species accepted and widely used throughout industry tor nonclinical studies of developmental toxicity (embryo-fetal toxicity/teratogenicity); 2) this strain has been demonstrated to be sensitive to developmental toxins; and 3) historical data and experience exist at the Testing Facility(1 ·3l_Number:Initial population acclimated: 60 virgin female rats. Population selected for study: 40 mated female rats (8 per dosage group).

Body Weight and Age:

Female rats will be ordered to have body weights of 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights recorded the day after receipt will be documented in the raw data.

Sex:

Female rats will be given the test substance. Male rats of the same source and strain will be used only as breeders and are not considered part of the Test System.

Source:

Charles River Laboratories, Inc. 4l8-023P:PAGE 47 Protocol 418-023P. The rats will be shipped in filtered cartons by air freight and/or truck from Charles River Laboratories, Inc., to the Testing Facility.

Identification:

Rats are permanently identified using Mone!® self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male rats are given unique permanent identification numbers upon assignment to the Testing Facility's breeder male rat population. Female rats are assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study on the basis of day 0 of presumed gestation body weights.

RANDOMIZATION AND COHABITATION:

Upon arrival, male and female rats will be assigned to individual housing on the basis of computer-generated random units. After acclimation, virgin female rats will be cohabited with breeder male rats, one male rat per female rat. The cohabitation period will consist of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day O of presumed gestation and assigned to individual housing. Healthy mated female rats will be assigned to dosage groups based on computergenerated (weight-ordered) randomization procedures.

ADMINISTRATION:

Route and Reason for Choice: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure.

Method and Frequency: Female rats will be given the test substance once daily on days 6 through 20 of presumed gestation. Dosages will be adjusted daily for body weight changes and given at approximately the same time each day.

Rationale for Dosage Selection:

Dosages were selected on the basis of a 28-day (gavage) study of T-7485 in rats (Redfield Laboratories Study Number 132-007) with an approximate 14-day recovery period which resulted in no mortality or abnormal clinical observations as well as no changes in body weights, body weight changes, feed consumption, peripheral neuropathy, motor activity, audio/visual response, hematology, clinical chemistry, gross pathology and histopathology. The administration of 900 mg/kg/day T-7485 resulted in significant increases in male liver and female kidney weights when compared to control animals and establishes the no-observable-adverse-effect-level (NOAEL) at 300 mg/kg/day T-7485. The highest dosage level selected should induce some overt developmental and/or maternal mortality. The intermediate dosage levels should produce minimal observed toxic effects. The lowest dosage level should not produce any evidence of either maternal or developmental toxicity.

Dosage Levels, Concentrations and Volumes:

Number of Dosage Concentration Volume

Grouo Rats (mg/kg/dav) (mQ/ml) (mlll<Q) Arous Batch Number

I 8 0 0 10 8-418-023P-A(Day.Month. Year)

II 8 100 10 10 8-418-023P-8(0ay.Month. Year)

Ill 8 300 30 10 8-418-023P-C(Oay.Month.Year)

IV 8 1000 100 10 8-418·023P-D(Day.Month. Year)

V 8 2000 200 10 8-418-023P-E(Day.Month.Year)

The test substance will be considered 100% pure for the purpose of dosage calculations.

TESTS, ANALYSES AND MEASUREMENTS:

Viability:

All Periods: At least twice daily.

Clinical Observations and/or General Appearance:

Acclimation Period: Weekly

Predosage Period: Day O of presumed gestation.

Dosage Period: Daily before dosage. Postdosage obseNations will be recorded approximately 60± 10 minutes after administration.

Day of Sacrifice:Once

Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director and/or Study Monitor.

Body Weights:

Acclimation Period: Weekly

Predosage Period: Day O of presumed gestation

Dosage Period: Daily

Day of Sacrifice: Prior to sacrifice

Feed Consumption Values (recorded and tabulated):

Predosage Period: Day O of presumed gestation.

Dosage Period:Days 6, 9 12, 15, 18 and 21 of presumed gestation

Day of Sacrifice: Feed left recorded.

Feed consumption values may be recorded more frequently if it is necessary to replenish the feed. These intervals will not be tabulated.

Mating Performance:

Mating will be evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ.

Caesarean-Sectioning Observations:

Rats will be Caesarean-sectioned on day 21 of presumed gestation. To minimize bias, Caesarean-sectioning and subsequent fetal observations will be conducted without knowledge of dosage group. The gravid uterus will be excised and weighed. The fetuses will be removed from the uterus and placed in individual containers. The rats will be examined for number and distribution of:

Corpora Lutea

Implantation Sites

Live and Dead Fetuses (A live fetus is defined as one that responds to stimuli; a dead fetus is defined as a term fetus that does not respond to stimuli and that is not markedly autolyzed; dead fetuses demonstrating marked to extreme autolysis are considered to be late resorptions.)

Early and Late Resorptions (A conceptus is defined as a late resorption if it is grossly evident that organogenesis has occurred; if this is not the case, the conceptus is defined as an early resorption.)

Fetal Observations:

To minimize bias, fetal observations will be conducted without knowledge of dosage group.Fetuses will be examined for sex and for gross external alterations. Late resorptions and dead fetuses will be examined for gross external alterations to the extent possible. The body weight of each fetus will be recorded. Only body weights of live fetuses will be used to determine litter fetal body weight averages. Fetuses with gross external alterations will be fixed in Bouin's solution; all other fetuses will be discarded. Representative photographs of fetal gross external alterations will be taken.

METHOD OF SACRIFICE:

Rats will be sacrificed by carbon dioxide asphyxiation. Live fetuses will be sacrificed by an intraperitoneal injection of Beuthanasia®-D Special, manufactured by Schering Plough Animal Health.

NECROPSY:

Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation. Unless specifically cited below, all other tissues will be discarded.

Scheduled Sacrifice:

On day 21 of presumed gestation, female rats will be Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The gravid uterus will be excised and weighed. Uteri of apparently nonpregnant rats will be examined while being pressed between glass plates to confirm the absence of implantation sites.

Rats Found Dead or Moribund:

Rats that die or are sacrificed because of moribund condition, abortion or premature delivery will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Pregnancy status and uterine contents of female rats will be recorded. Gravid uterine weights will not be recorded if precluded by autolysis. Aborted fetuses, delivered pups and/or concepti in uteri will be examined to the extent possible, using the methods described above. Uteri of apparently nonpregnant rats will be examined while being pressed between glass plates to confirm the absence of implantation sites.

STATISTICAL EVALUATION:

Averages and percentages will be calculated. Litter values will be used where appropriate. Additional procedures and/or analyses may be performed if deemed appropriate."

Red Perioral Substance

Endpoint Details

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