"2. 7. Methods

2.7.1. Dosage Administration

2.7.2. Rationale for Dosage Selection

Dosages were selected on the basis of a 28-day (gavage) study of T-7 485 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 was expected to produce some toxicity. The intermediate dosage levels were expected to produce minimal observable toxic effects, while the lowest dosage level was expected to produce no evidence of either systemic or reproductive toxicity [no-observable-adverse-effect level (NOAEL)].

2.7.3. Route and Rationale for Route of Administration

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.

2.7.4. Frequency of Administration

2.7.4.1. P Generation Rats Male and female rats were given the test substance or vehicle once daily, beginning at approximately six weeks of age (at least 70 days before cohabitation) and continuing until the day before sacrifice. Dosages were adjusted based on the most recently recorded body weight3 and given at approximately the same time each day. Dams in the process of delivering pups were not dosed until completion of parturition, in order to preclude possible disruption of maternal behavior and/or cannibalization of the pups. Consequently, some darns were not administered one daily dosage during the delivery period. No dam missed more than one daily dose.

2. 7 .4.2. Fl Generation Rats Fl generation pups may have been exposed to the test substance during maternal gestation (in utero exposure) or via maternal milk during the postpartum period. Fl generation rats were given the same dosage level of the test substance as their respective P generation sires and dams. Dosages were given once daily, beginning at weaning (approximately 70 days before cohabitation) and continuing until the day before sacrifice. Dosages were adjusted at least wt:ekly for body weight changes and given at approximately the same time each dayb. Dams in the process of delivering pups were not dosed until completion of parturition, in order to preclude possible disruption of maternal behavior and/or cannibalization of the pups. Consequently, some dams were not administered one daily dosage during the delivery period. No dam missed more than one daily dose.

2. 7 .4.3. F2 Generation Pups

F2 generation pups may have been exposed to the test substance during maternal gestation (in utero exposure) or via maternal milk during the postpartum period.

2.7.5. Method of Study Performance

2.7.5.1. P Generation Rats

The P generation rats were observed for viability at least twice each day of the study and for general appearance weekly during the acclimation period. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths before and approximately 60 ± 10 minutes after dosage administrationa. Body weights of the male rats were recorded weekly during the acclimation and dosage periods and on the day of sacrifice. Body weights of the female rats were recorded weekly during the acclimation, precohabitation and cohabitation periods, on DGs 0, 7, 10, 14, 18, 21 and 25 (if necessary) and on DLs 1, 5, 8, 11, 15 and 22 (terminal body weight). Feed consumption values for the male rats were recorded weekly during the acdimation and dosage periods. Feed consumption values for the female rats were recorded weekly during the acclimation and precohabitation periods, on DGs 0, 7, IO, 14, 18, 21 and 25 (if necessary) and on DLs 1, 5, 8, 11, and 15. During cohabitation, when two rats occupied the same cage with one feed jar, replenishment of feed jars was documented but individual values were not recorded or tabulated. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15. Estrous cycling was evaluated daily by examination of vaginal cytology beginning 21 days before the scheduled cohabitation period and continuing until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. On the day of scheduled sacrifice, an examination of vaginal cytology was performed to determine the stage of estrous cycleb. Within each dosage group, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug in situ were considered to be at DG O and assigned to individual housing. If a female rat had not been mated after two weeks, the pair of rats was separated without further opportunity for mating. The P generation female rats were observed for duration of gestation (DG O to the day the first pup was observed), fertility index (percentage of matings that result in pregnancy), gestation index (percentage of pregnancies that result in birth of live litters), number and sex of offspring per litter (live and dead pups), number of implantation sites, general condition of the dam and litter during the postpartum period, litter size and viability (tabulated on DLs 1, 5, 8, 15 and 22), viability indices (percentages of pups born that survive to DLs 1 and 5), lactation index (percentage of pups that survive from DLs 5 to 22) and percent survival and sex ratio (tabulated on DLs 1, 5, 8, 15 and 22). Maternal behavior of the P generation dams was recorded on DLs 1, 5, 8, 15 and 22a. Deviations from expected maternal behavior were recorded, when and if observed, on all other days of the postpartum period.

2. 7 .5.2. Fl Generation Pups/Litters

Litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross external alterations. DL 1 was defined as the day of birth and was ~so the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Each litter was evaluated for viability at least twice each day of the 22-day postpartum period. Dead pups observed at these times were removed from the nesting box. Fl generation pups in each litter were counted once daily. Physical signs (including variations from expected lactation behavior and gross external alterations) were recorded for the pups each day3 • Pup body weights were recorded on DLs 1 (livebom pups), 5, 8, 15 and 22.

2. 7 .5.3. Fl Generation Rats

The Fl generation rats were observed for viability at least twice each day of the study. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths before and approximately 60 ± 10 minutes after dosage administrationb. Body weights of the male rats were recorded weekly during the postweaning period and on the day of sacrifice. Body weights of the female rats were recorded weekly during the postweaning period to cohabitation, on DGs 0, 7, 10, 14, 18, 21 and 25 (if necessary) and on DLs 1, 5, 8, 11, 15 and 22 (terminal body weight). Feed consumption values for the male rats were recorded weekly during the po$tweaning period to cohabitation and weekly during the postcohabitation period until sacrifice. Feed consumption values for the female rats were recorded week:Jy during the postweaning period to cohabitation, on DGs 0, 7, 10, 14, 18, 21 and 25 (if necessary) and on DLs 1, 5, 8, 11, and 15c. During cohabitation, when two rats occupied the same cage with one feed jar, replenishment of feed jars was documented but individual values were not recorded or tabulated. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15. Female rats were evaluated for age of vaginal patency, beginning on day 24 postpartum. Male rats were evaluated for the age of preputial separation, beginning on day 35 postpartum. Body weight was recorded for each rat upon determination of sexual maturation. Estrous cycling was evaluated daily by examination of vaginal cytology beginning 21 days before the scheduled cohabitation period and continuing until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. On the day of scheduled sacrifice, an examination of vaginal cytology was performed to determine the stage of estrous cycle3 • A table of random units was used to assign Fl generation rats to cohabitation, one male rat per female rat. If random assignment to cohabitation resulted in the pairing of Fl generation siblings, an alternate assignment was made. The cohabitation period consisted of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug in situ were considered to be at DG O and assigned to individual housing. If a female rat had not been mated after two weeks, the pair of rats was separated without further opportunity for mating. The Fl generation female rats were observed for duration of gestation (DG O to the day the first pup was observed), fertility index (percentage of matings that result in pregnancy), gestation index (percentage of pregnancies that result in birth of live litters), number and sex of offspring per litter (live and dead pups), number of implantation sites, general condition of the darn and litter during the postpartum period, litter size and viability (tabulated on DLs 1, 5, 8, 15 and 22), viability indices (percentages of pups born that survive to DLs 1 and 5), lactation index (percentage of pups that survive from DLs 5 to 22) and percent survival and sex ratio (tabulated on DLs 1, 5, 8, 15 and 22). Maternal behavior of the Fl generation dams was recorded on DLs 1, 5, 8, 15 and 22b. Deviations from expected maternal behavior were recorded, when and if observed, on all other days of the postpartum period

2.7.5.4. F2 Generation Pups/Litters

Litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross external alterations. Each litter was evaluated for viability at least twice each day of the 22-day postpartum period. Dead pups observed at these times were removed from the nesting box. Fl generation pups in each litter were counted once daily. Physical signs (including variations from expected lactation behavior and gross external alterations) were recorded each day. Pup body weights were recorded on DLs 1 (liveborn pups), 5, 8, 15 and 22a.

2.7.6. Gross Necropsy

2. 7 .6.1. P and Fl Generation Rats

Adult P and Fl generation rats were sacrificed by carbon dioxide asphyxiation, necropsied and examined for gross lesions. Representative photographs of paternal and maternal gross lesions are available in the raw data. Gross lesions were retained in neutral buffered 10% formalin and examined histologically. Tissue trimming and histopathology were performed under the supervision of or by a Board Certified Veterinary Pathologist. Histopathology evaluations were conducted at Research Pathology Services, Inc., New Britain, Pennsylvania. The results are available in APPENDIXK. Gross necropsy included an initial physical examination of external surfaces and all orifices, as well as an "internal examination of tissues and organs in situ. The following were examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculo/skeletal carcass. The lungs of the F 1 generation male rats and P and Fl generation female rats were perfused with neutral buffered 10% formalin and the urinary bladder of postweaning rats was inflated with neutral buffered 10% formalinb. The following organs were individually weighed and organ-to-body weight and organ-tobrain weight ratios were calculated: brain, kidneys, spleen, ovaries, testes, thymus, liver, adrenal glands, pituitary, uterus with oviducts and cervix, left epididymis (whole and cauda), right epididymis, prostate and seminal vesicles (with coagulating glands and with and without fluidf The following tissues were retained in neutral buffered 10% formalin for possible histological evaluation: pituitary, liver, adrenal glands, kidneys, vagina, uterus with oviducts, cervix and ovaries, left testis (initially fixed in Bouin's solution for 48 to 96 hours), seminal vesicles (with coagulating gland), right epididymis and prostated. Histological examination was performed on tissues from ten randomly selected rats per sex from the control and high dosage groups. All gross lesions were examined histologically. In addition to gross lesions such as atrophy or tumors, testicular histopathological examination was conducted to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda (accomplished by evaluation of a longitudinal section) and conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium. Reproductive organs of the rats suspected of reduced fertility (e.g., those rats that failed to sire healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected) were subjected to histological evaluation3 • Rats that died or were sacrificed because of moribund condition were examined for the cause of death or moribund condition on the day the observation was made. The rats were examined for gross lesions. Representative photographs of gr_oss lesions are available in the raw data. Gross lesions were retained in neutral buffered 10% formalin and examined histologically. Tissues were weighed and retained and examined histologically as described for male rats sacrificed at the completion of the cohabitation period and female rats sacrificed on DL 22b.

2.7.6.1.1. Male Rats

All surviving male rats were sacrificed after completion of the cohabitation period days of study (DSs) 122 to 123 for the P generation rats and DPs 129 to 141 for the Fl generation rats. For motility assessment, the right vas deferens was excised and immediately placed in a petri dish containing 10 mL of I% Bovine Serum Albumin dissolved in Phosphate Buffered Saline. The solution was prewarmed at a temperature of approximately 38°C. Following a three minute period, a sample of sperm was collected using a 100 micron deep cannula and immediately loaded into a prewarmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields were selected and stored on an optical disk. These images were analyzed for percent motility. The right epididymis was removed, weighed and divided in half by cross sectioning at the middle. The cauda and distal midsection were placed on dry ice for analysis for total sperm count evaluations. This frozen section of the epididymis was then transferred to Pathology Associates, Frederick, Maryland, and stored frozen at -70°C until evaluation for total sperm count. If a unilateral gross lesion was observed in the right epididymis, then the left epididymis was used for sperm analysis and treated as described above for the right epididymis. Each frozen epididymis was thawed and the caudal section trimmed, weighed (weight utilized for spem1 analysis only) and homogenized. A 100 microliter sample of the suspension was stained with a dye which uniquely stains the and loaded into the analyzer. Twenty fields per rat were counted and reported adjusted for caudal epididymidal weight. Two slides of sperm per rat were stained with Eosin for sperm morphology assessment. A minimum of 200 sperm cells per rat were evaluated for morphological development. See APPENDIX L for the results of all spe1m evaluation parameters (motility, sperm and spermatid count and morphology). The right testis was removed from each rat and placed on dry ice for analysis of testicular spermatid counta. Toe frozen testes were transferred to Pathology Associates, Frederick, Maryland, and stored frozen at -70°C until evaluation for testicular sperm count. Each frozen testis was removed from the freezer, thawed, the tunica removed, the testicular parenchyma weighed and homogenized. A 100 micro liter sample of the suspension was stained with a dye which uniquely stains the sperm heads. A sample of the stained sperm was placed in a 20 micron deep glass chamber and loaded into the IVOS automated sperm analyzer. Twenty fields per rat were counted and reported, adjusted for testicular weight.

2.7 .6.1.2. Female Rats

All surviving female rats were sacrificed on DL 22. The number and distribution of implantation sites were recorded. Rats that did not deliver a litter were sacrificed on DG 25 and examined for pregnancy status. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Female rats without a confirmed mating date that did not deliver a litter were sacrificed on an estimated DG 25. Dams with no surviving pups were sacrificed after the last pup was found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Gross lesions were retained in neutral buffered 10% formalin for possible histological evaluation.

2.7.6.2. Fl and F2 Generation Pups

Pups that died before initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered livebom and to have died shortly after birth. Pups were necropsied. When postmortem autolysis precluded these evaluations, it was noted in the necropsy data. Pups found dead on DLs 2 through 22 were necropsied and examined for gross lesions and the cause of death. When postmortem autolysis precluded these evaluations, it was noted in the necropsy data. All pups culled on DL 22 were sacrificed by carbon dioxide asphyxiation. The urinary bladder and lungs of all Fl generation pups were inflated with neutral buffered 10% formalina. Gross lesions were retained in neutral buffered 10% formalin for possible histological evaluation. Representative photographs of gross lesions are available in the raw data. Three pups per sex per litter, when possible, were randomly selected and examined for gross lesionsb. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. The following organs were weighed from the first randomly selected pup per sex per litter: brain, spleen, liver, thymus and kidneys. The following tissues were collected from three randomly selected pups per sex per litter and retained in neutral buffered 10% formalin for possible histological evaluation: kidneys, liver and gross lesions. All remaining pups were discarded without further examination.

2.7.7. Data Collection and Statistical Analyses

Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System and the Hamilton Thome IVOS. All data were tabulated, summarized and statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel [part of Microsoft Office 97 (version SR-2)] and/or The SAS System (version 6.12)."

Xiphoid, Small and Misshapen

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