RACB_Sample_Study Final.pdf

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SAMPLE STUDY ASSIGNMENT #1

STUDY TITLE: REPRODUCTIVE ASSESSMENT BY CONTINUOUS BREEDING

(RACB) IN HARLAN SPRAGUE DAWLEY RATS VIA DOSED FEED

FOR DIMORPHOLINODIETHYL ETHER (CAS # 6425-39-4)

1) Introduction

The Reproductive Assessment by Continuous Breeding study (RACB) is a multigeneration reproduction study composed of several phases: (1) Prestart chemistry and dose range-finding studies; (2) Treatment and cohabitation of F0 males and females to produce an F1 generation for assessment; (3) A crossover mating of F0 treated males and females with non-treated naive females and males to determine the affected sex; (4) cohabitation of F1 males and females to produce an F2 generation for assessment.

The test material for this study is dimorpholinodiethyl ether. This compound is a blowing agent for the production of flexible, molded, and moisture-cured foams and coatings.

2) Project Management

The work within this Study Assignment shall be conducted in Phases. Refer to the SOW for Project Management information and government-supplied materials.

[Note: The offeror shall provide a technical approach and a separate cost estimate for each phase.]

Phase 1: Prestart Chemistry: The contractor shall perform those work activities associated with bulk chemical analysis, validation of dose formulation/formulation analysis methods, and address health and safety concerns before animals are dosed.

Phase 2: Dose Range-finding: A Two-week Dose Range-finding study with males and non-pregnant females shall be conducted, followed by a second range-finding study using male and female rats treated during mating, gestation and lactation.

Phase 3: F0 production of F1 generation in definitive RACB assessment.

Phase 4: Assessment of the affected gender using a cross over mating test.

Phase 5. F1 production of F2 generation in definitive RACB assessment.

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3) Objectives

Conduct a two-week range finding study in non-pregnant animals and an RACB range-finding study in pregnant animals using doses specified by the NTP.

Conduct a definitive RACB study for this chemical using doses specified by the

NTP.

4) Test Material

Test material, dimorpholinodiethyl ether, CAS # 6425-39-4, shall be provided by the NTP, along with methods for bulk analysis, formulation of the test material in the diet, and analysis of the test material in the diet.

5) Vehicle

Test material shall be administered via the diet, NIH-07 Open Formula Certified Rat/Mouse Diet (Zeigler Brothers, Inc Gardners, PA, certified and irradiated).

6) Pre-Start Chemistry

Receive the test material and perform bulk chemical analysis (identity and purity confirmation) according to the procedures provided. Results shall be reported to the Contracting Officer’s Representative (COR) by telephone and e-mail as soon as they are available.

Conduct dose formulation analysis method performance evaluation, Prepare and analyze dose formulations according to the instructions provided by the NTP.

Provide a pre-start chemistry report. The pre-start chemistry report shall include:

1) a summary of the NTP-provided information on the source of the bulk chemical, storage requirements, identity and purity data,

2) confirmatory data on identity, purity (including representative chromatograms and spectra of the test article and reference material); provide copies of the SOPs for the bulk chemical reanalysis methods,

3) data on preparation, handling, homogeneity, and analysis of dose formulations as well as SOPs,

4) dose formulation analysis method performance evaluation procedures and validation data, including representative chromatograms or spectra,

5) health and safety procedures to be followed when using dimorpholinodiethyl ether.

Animals shall not be dosed until this report has been received by the NTP.

7) Animal Husbandry and General information

Test Species and Strain/Stock: Male and female Harlan Sprague Dawley rats from an NTP designated Harlan Laboratories facility.

Diet and Water: Water and NIH-07 feed (meal) shall be provided ad libitum. Feed shall be stored at 70°F or lower, 50% humidity or lower, and in a well-ventilated area stored raised above the floor. Batch numbers for feed and use period shall be

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recorded. Feed shall be used for no more than 120 days post milling. Laboratories must demonstrate that drinking water provided for animal use meets US/EPA National Primary Drinking Water Regulations (analysis at least once/year).

Bedding: Sani-Chips cage litter (P.J. Murphy Forest Products, Montville, NJ) shall be used throughout the study. Analyses of the bedding and certification of irradiation shall be provided by the Supplier for each lot/batch, and copies shall be maintained in the study records.

Environment: The light cycle in the animal rooms shall be twelve-hour light/dark.

The temperature of the animal room shall be maintained at 72 + 3ºF. The relative humidity of the animal room shall be maintained at 35 – 65%. If the temperature or humidity falls or rises out of range, correction shall be made within two hours. The ventilation system shall provide a minimum of 10 air changes of room air per hour without drafts. There shall be no recirculation of room air unless it has been treated to remove all particulates and toxic vapors by effective filters to avoid the recirculation of contaminants.

Housing: Solid-bottom clear polycarbonate cages shall be used. Animals shall be singly housed except during mating and lactation periods. During mating periods, one male is housed with one female (except for dominant lethal tests where one male is housed with two females). For mating, the female is introduced into the male’s cage in the early afternoon. During lactation periods, dams shall be housed with their litters.

Pre-study quarantine and acclimation period: Animals shall be held for a 10-14 day quarantine and acclimation period prior to study start and observed for general health status by the Laboratory Animal Veterinarian. During this time animals are permanently identified. At the end of the quarantine/acclimation period the animals shall be examined and released from quarantine (if healthy) for study by the Laboratory Animal Veterinarian.

Sentinel and Disease Screening Animals: Extra animals shall be included in shipment of animals to be used for disease screening and as sentinels for studies over 6 weeks in duration. Of these animals, upon arrival at the laboratory, 5 males and 5 females, shall be examined for disease and parasites by the Laboratory Animal Veterinarian and/or the Veterinary Pathologist. Diagnosis of lesions seen at necropsy of these animals shall be confirmed by histopathologic evaluation and/or microbiological culture. Serum shall be obtained and prepared for analysis for pathogens. Specifically, serum shall be prepared, shipped and analyzed as follows:

Diluted serum in at least 100 ul samples (1 part serum: 5 parts physiological saline) shall be stored at 4°C or colder until shipped on wet ice to an NTP-designated laboratory (RADIL, Univ. of MO, 1600 E. Rollins, Columbia, MO 65211 for their clinical rat standard profile) for antibodies. Rats shall be tested for Pneumoncystis carinii at the 6 month time point.

Disease screening is conducted on animals during the initial quarantine period at the laboratory, 4 weeks later and at least every 6 months, and at termination. Animals (5 males and 5 females per timept) at the initial 4 wk and termination timepts shall be bled and necropsied. At other timepts, animals shall be bled only. Details for disease screening will be included in chemical specific Study Assignments. For

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sentinel/disease screening animals, body weights and food consumption shall not be measured during the study.

8) Two-week Dose Range-finding study with Males and Non-Pregnant Females (Phase 2a)

In this dose range-finding study, animals shall be dosed via the diet for 14 consecutive days.

Animals shall be sexually mature at the start of dosing, approximately 11 weeks of age for females and 13 weeks of age for males. Test material shall be provided via the diet for the duration of the study at dose levels of 0, 100, 1,000, 5,000 and 10,000 and 20,000 ppm.

Five animals per sex per dose group shall be used.

Individual body weights shall be determined 1-2 days prior to study start for randomization of the animals into groups with body weights as close as possible and no more than + 10% from the mean body weight (by sex). During treatment, body weights shall be determined twice a week including the first and last days of dosing (study days 1, 4, 7, 11 and 14). Food consumption shall be recorded twice a week at intervals coinciding with body weight measurements.

Animals shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart, before 10:00 am and after 2:00 pm, including weekends and holidays, for moribundity/mortality. Formal clinical signs (out of cage observations) shall be recorded for individual animals daily. Signs of toxicity detected at times other than a formal clinical observation time shall be noted and recorded.

At study termination, animals shall be given an external and internal examination for gross lesions. The following organs shall be weighed: liver, thymus, left and right kidney, left and right testes, epididymides, seminal vesicles with coagulating glands, and prostate. These organs and gross lesions shall be saved in formalin.

Process testes to slides and examine histopathologically.

Results for survival, clinical signs, body weight, food consumption, organ weights (absolute and relative), and gross necropsy findings shall be provided to the COR within 5 days of final necropsy. Histopathology data shall be provided within 4 weeks of necropsy. This information will be used by the NTP to choose doses for the following range-finding study using pregnant dams.

9) RACB Range-finding Study (Phase 2b)

In this range-finding study, males and females shall be dosed continuously via the diet for a 7-day pre-breeding period, through mating, gestation, birth and lactation until PND 4 at which time the study shall be terminated. See Table 1 for number of animals per group. At start of dosing, females shall be 10 weeks of age and males shall be 12 weeks of age. For the purpose of responding to this RFP, assume dose levels of 0, 100, 1,000, 5,000 and 10,000 ppm.

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Table 1: Number of Harlan Sprague Dawley Parental rats for the RACB Dose Range-finding Study.

Study Males Females Exposure Total Animals

Range Finding

Group 1 (Control)

14 14

Exposure starts 7 days prior to mating and continues through PND 4. An interim sacrifice of 3 dams from the control, low and high dose groups shall be performed on GD 18 and PND 4 for sample collection.

Group 2 (100 ppm)

14 14 28

Group 3 (1,000 ppm)

8 8 16

Group 4 (5,000 ppm)

8 8 16

Group 5 (10,000)

14 14 28

Animals shall be randomly assigned to one of four (4) treatment groups and one control group as in Table 1 so that each group has comparable body weights, within + 10% of the mean body weight by sex at initiation of dosing (if it is necessary to use a few animals outside the range, approval of the COR shall be obtained).

Body weights shall be measured 1-2 days prior to start of dosing for randomization and on the first day of dosing. Rats shall be dosed for one week (7 days) prior to mating, and throughout the mating, gestation and lactation periods.

After the 7 day pre-mating period, rats shall be randomly assigned within each treatment group to mating pairs of one male and one female per cage. These pairs shall be cohabitated for up to 15 days or until dams are sperm positive, whichever comes first. Mating activity shall be ascertained each morning by in situ plug or the presence of sperm after vaginal lavage. The day vaginal sperm are confirmed is designated GD 0.

After the mating, breeding pairs shall be separated in individual housing and dams allowed to deliver offspring. Females not found to be sperm positive during the cohabitation period shall be individually housed after day 15 of cohabitation and monitored for delivery of litters. Day of delivery is PND 0.

Animals shall be observed for mortality and moribund status twice daily, once before 10 am in the morning and once after 2 pm in the afternoon, at least 6 hours apart.

Formal clinical observations shall be conducted and recorded once daily.

Body weights for males shall be taken twice a week during the study (study days 1, 4, 7, 11, 14, etc).

Body weights for the females shall be taken twice per week during the pre-mating period; twice a week during the mating period while sperm-negative and daily if sperm positive for GD0 through PND4. Body weight changes shall be calculated for the intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21 and PND 1-4.

Feed consumption measurements shall be recorded for all animals twice per week during the 7-day pre-mating exposure period (at intervals which coincide with body

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weight measurements). Consumption measurements shall be suspended during mating; and resumed for males after separation, and for females for the intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, and PND 1-4.

Upon delivery, pups shall be counted, gender determined, weighed and examined grossly for external physical abnormalities as soon as possible on the day of birth and also on PND 4. Weights shall be taken by gender by litter. Anogenital distance shall be measured on PND 1 along with individual pup weight.

The following data shall be reported:

Adult clinical signs, body weights and feed consumption, Number pregnant females, pre-coital interval, day of delivery, number of live and dead pups delivered per litter, pup body weights on PND 0, 1 and 4, AGD on PND 1 with corresponding body weight, and pup survival through PND 4.

Euthanize adults and pups on PND4.

Biological Sample collection:

GD 18: Use 3 randomly selected pregnant dams from each the control, high and low dose groups to provide tissue for possible later analysis by an NTP designated laboratory (for test material concentration). Using the sperm positive finding in females to determine GD 0, sacrifice these dams on GD 18 and collect maternal plasma (approximately 3.0 ml), amniotic fluid (pooled by litter, approximately 250 ul), and fetuses (whole, pooled by sex by litter). (If not all females in a group appear to be pregnant, consult the COR to determine if biological sample collection should proceed.

PND 4: From 3 randomly selected dams and their litters of each the control, high and low dose groups, collect maternal plasma (3.0 ml) from these 3 dams; and collect one male and one female pup (whole pup) randomly selected from each of their litters at PND 4.

Samples from the GD 18 and PND 4 tissue collection are to be stored at -20°C and shipped to an NTP designated location as instructed by the COR. Samples must be identified so that pup samples can be correlated with their dam.

10) RACB Definitive Study Design

Overview

In this study, males and females shall be dosed continuously for the duration of the study via the diet. Dosing shall start 14 days before mating. At the start of dosing, females shall be 9 weeks of age and males shall be 11 weeks of age. At the start of F0 mating, animals shall be sexually mature, 11 weeks of age for females and 13 weeks of age for males. F0 parental animals shall be mated (lifetime pairs) and produce 3 litters (F1a, F1b and F1c). The F1a and F1b litters shall be evaluated through PND 4. The F1c litter shall be evaluated through PND 21 and a subset of the F1c animals shall be continued on study to produce the F2 generation. After the

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F1c animals are 13 weeks of age, they shall be mated 3 times to produce the F2a, F2b and F2c litters.

F0 mating and F1 assessment (Phase 3) At the start of dosing, females shall be 9 weeks of age and males shall be 11 weeks of age. Animals (25 male and 25 female animals per group, aiming to obtain at least 20 pregnant females per group) shall be randomly assigned by stratified body weight to one of three (3) treatment groups and one control group (Table 2) so that each group has comparable body weights, within + 10% of the mean body weight by sex at study initiation. Sentinels shall also be assigned (15/sex). For the purposes of responding to this RFP, assume doses shall be 0, 50, 500 and 2,500 ppm via diet.

Dosing shall begin 14 days before mating. Males and females are to be weighed 1-2 days prior to dosing for randomization and on Day 1 of dosing.

Body weights and feed consumption shall be measured twice weekly (study days 1, 4, 7, 11, 14 etc) for males and non-pregnant and non-lactating females. Body weights for the females shall be taken twice per week during the pre-breeding period;

twice a week during the mating period if sperm-negative and daily if sperm positive for GD0 through PND4. Body weight changes shall be calculated for the intervals:

GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21 and PND 1-4, 4-7, 7-10, 10-13, 13- 16, 16-19 and 19-21 if sperm-positive. (Day of finding of sperm positive = GD0.)

Throughout the study, the Contractor shall make twice-daily (7 days per week) cage side inspections for clinical signs and/or symptoms as well as to determine the delivery status of pregnant females. These checks shall take place before 10 am and after 2 pm daily, at least 6 hours apart. During periods of delivery, the frequency of checks shall be increased. Formal clinical observations shall be made once per day and findings for individual animals recorded.

Table 2: Number of Harlan Sprague Dawley Parental F0 rats for the Definitive Study.

Study Males Females Exposure Total Animals

Definitive RACB

Group 1 (Control)

25 25

Exposure starts 2 weeks prior to mating of F0 adults and continues through PND 21 of the F2c litter.

Group 2 (50 ppm)

25 25 50

Group 3 (500 ppm)

25 25 50

Group 4 (2,500 ppm)

25 25 50

Fourteen days after the start of dosing, the contractor shall randomly pair the animals, one male and one female per cage, from the same treatment group. These pairs shall be cohabitated for up to 15 days or until dams are sperm positive, whichever comes first. Mating activity shall be ascertained each morning by in situ plug or the presence of sperm after vaginal lavage. The day of finding sperm positive

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shall be noted as GD0 and used to calculate the time to achieving pregnancy (pre-coital interval).

Males and females shall be separated upon the finding of sperm positive, allowing pregnant females to deliver their pups. Females not found to be sperm positive during the cohabitation period shall be individually housed after day 15 of cohabitation and monitored for delivery of pups. Dams that do not deliver pups shall be examined internally on expected GD24 (39 days after start of cohabitation) for signs of implantation loss.

Pups shall be evaluated from PND 0 to PND 4 as below. Two days after PND 4, each pair shall be re-established and the sequence repeated for two more litters (F1b, F1c). After breeding for the third litter (F1c), the F0 pairs shall be permanently separated and individually housed. The separated F0 sexes shall continue to be dosed and body weights and food consumption collected weekly until the day of F0 necropsy (which occurs when matings are complete, litters are weaned and no further evaluations are stipulated by the protocol or requested by the COR, such as crossover matings). (For costing purposes, a cross-over mating shall be required in this study).

The following data from each dam/litter (F1a, F1b, F1c) shall be collected:

1) Pre-coital interval and length of gestation

2) Number of litters per pair

3) Number of pairs with no litters

4) On PND 0, number of live and dead pups

5) On PND 0, sex ratio and weight by sex for each litter

6) On PND 1, anogenital distance (AGD) and corresponding pup body weight.

7) On PND 4, number of pups, sex ratio and weight by sex for each litter (pre-cull for F1c).

8) Presence and type of developmental anomalies in the pups (PND 0-4).

9) Calculated mean body weight of pups (by sex per litter, and total per litter) and percent live and dead pups for PND 0, 1 and 4.

An interim summary shall be submitted on F1 litter size, weight and sex distribution for each litter (i.e., F1a, F1b, and F1c) within 5 days after the last expected delivery for that littering.

Pups from the F1a and F1b litters shall be euthanized on PND 4 without further evaluation. The last litter (F1c) shall be retained and maintained on dosed feed to produce the F2 generations.

F1 Maturation

1) All F1c pups shall be maintained until PND 4, at which time the F1c litters shall be culled to 10 offspring per litter, ideally as close as possible to 5 males and 5 females per litter; if there are fewer than 5 pups of one sex in a litter, keep all pups of that sex in that litter; if there are fewer than 10 pups in a litter, all pups in that litter shall be kept. Litters shall be weaned on PND 21;

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2) Count and individually weigh F1c offspring on PND 4 (after cull), 7, 14, and 21, and calculate percent survival for each time;

3) Collect body weight data on dams of F1c pups for PND 4, 7, 14, and 21;

4) Evaluate the F1c males for the presence of areolae (nipples) on PND 13;

5) Evaluate the F1c males for testicular descent (daily starting approximately

PND14 until attainment), and for prepuce separation (daily starting approximately PND 35 until attainment);

6) Evaluate F1c females for the first day of vaginal opening (daily starting approximately PND 23 until attainment).

7) Body weights shall be taken on day of attainment of preputial separation and vaginal opening.

8) At approximately 75 days of age, begin daily vaginal lavage for 16 days for estrus cyclicity evaluation of all females

9) Collect weekly measurements of body weights after weaning.

F0 Determination of Affected Sex (Phase 4):

A reduction in fertility has been attributed to test article exposure (a statistically significant decrease or a decrease of 2 or more pups per litter); the Contractor shall conduct a cross-over mating. A cross-over mating shall consist of (1) mating all the high dose males with non-dosed naive females; and (2) mating all high dose females with non-dosed males. Exposure to test article by feed shall be suspended for the 7-day cohabitation mating period. Non-treated naïve females and males shall be purchased in a number sufficient for there to be one-to-one mating pairs. Non-treated animals shall be mated with the high dose males and females when they are sexually mature, 11 weeks old for females and 13 weeks old for males. Mating pairs, one male and one female, shall be cohabitated until evidence of mating (vaginal plug or vaginal sperm) is found or 7 days, whichever comes first. Upon evidence of mating or after 7 days if there is no evidence of mating, males and females shall be housed separately and treated animals shall be again placed on test article-containing feed. Dams shall be allowed to deliver their litters. Litters from the cross-over mating shall be evaluated through PND 4. The following data shall be collected:

number of females with evidence of mating, number of females delivering a litter, number and sex of live and dead pups on PND 0, 1 and 4. Dams and pups shall be terminated on PND 4. Non-treated males used for cross-over matings shall be terminated after the cross-over mating period. The treated males (as well as vehicle control males from the RACB portion of the study) shall not be terminated until it is determined that no further testing of them (such as dominant lethal test) is required.

If a substantial male contribution to the adverse reproductive effect is indicated by the cross-over mating, a male dominant lethal test may be conducted. If required, the dominant lethal test shall be conducted as soon as possible following completion of the crossover mating. (For costing purposes, assume a dominant lethal test shall be required.) The dominant lethal test uses all the F0 treated and vehicle control males which are mated to naïve females. A sufficient number of naive females must be purchased so as to have two females to breed with each male.

Females shall be provided vehicle control feed and shall be 11 weeks old for mating.

Females shall be evaluated for the presence of vaginal sperm or a sperm plug daily for the 5 day mating period. Females shall be separated from the males after evidence of mating is found or after 5 days, whichever comes first. During the cohabitation period, dosing of males shall be suspended. Females shall

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be sacrificed in late gestation (GD19) for analysis of uterine contents for total implantation sites, live fetuses, resorption sites, and dead embryos and fetuses.

Corpora lutea shall also be counted.

(Continuation of Phase 3) F0 Female Vaginal Smears prior to Necropsy

For the F0 females, vaginal smears shall be collected and evaluated for 16 days prior to necropsy.

F0 Necropsy

Necropsy F0 animals after completion of any crossover mating/dominant lethal study, F1 offspring have been weaned, and vaginal cytology assessment of F0 females is complete. The necropsy includes an external examination of body orifices with special attention paid to the genitourinary system, examination of the internal organs and weighing and fixation of selected organs/tissues as described below. At F0 necropsy, the following shall be collected/conducted:

Left cauda epididymal spermatozoa measurements: number, density, and motility.

Left testicular measurements: spermatid head count.

Collect weights on:

Whole body Liver R & L Testis R & L Kidneys R & L Epididymis Thyroid gland (fixed weight) R & L Cauda epididymis R & L Ovaries Seminal vesicles (w/ Coagulating Glands) Thymus Levator Ani Bulbocavernosus Muscle Complex (if triggered) (assume triggered for costing purposes)

Paired Cowper’s glands (if triggered) (assume triggered for costing purposes)

Dorsolateral & ventral prostate (separately)

Fix the following Tissues:

Vagina, cervix, and uterus (together) Ovaries Right Testis and epididymis Thyroid gland Dorsolateral and ventral prostate Liver (if triggered) Seminal vesicles (w/ coagulating glands attached) Kidney (if triggered) Paired Cowper’s glands (if triggered) Adrenal Levator Ani Bulbocavernosus Muscle Complex (if triggered)

Pituitary

Retained Nipples (when present, one from each male)

Any abnormal organs (assume none for costing purposes)

Any target organs (assume none additional for costing purposes)

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For organ weights, report as absolute and relative to body weight.

Fix ovaries, testes and epididymides in modified Davidson’s fixative. Fix other tissues in neutral buffered formalin.

For costing purposes, assume liver and kidney are target tissues.

Therefore, these organs shall be weighed, fixed in NBF and examined histopathologically.

The Cowpers’s (bulbourethral) glands and levator ani bulbocavernosus (LACB) muscle complex will be grossly examined in all necropsied animals. However, they will be collected, fixed and examined histologically only if there are other lesions, malformations, or abnormalities consistent with anti-androgenic activity.

(For costing purposes, assume these are triggered tissues.)

Prepare slides for all protocol-required tissues for all dose groups (H & E). Perform histopathologic evaluation of these tissues in the high dose and control groups. If treatment-related findings are seen at the high dose for a certain tissue, then all dose levels for that tissue shall be examined. For costing purposes, assume that microscopic treatment-related findings are seen in testes and epididymides from high dose animals, triggering the evaluation of all male reproductive tissues at all dose levels.

F1c offspring assignment:

When F1c offspring are approximately 9-10 weeks of age and measurements for attainment of sexual development are completed, 2 males and 2 females per litter shall be selected to produce the F2 generation. Before mating, all females shall be assessed for estrus cycling for approximately 16 days.

Non-selected males and females (3/sex/litter) shall be necropsied at approximately 13 weeks of age and assessed the same as the F0 adults. Non-selected males shall be evaluated for the presence of nipples on shaved abdomens on the day prior or the day of sacrifice (if nipples were found on PND 13). If nipples are found, one nipple per male shall be collected and processed for paraffin sectioning and evaluated for mammary tissue confirmation.

Prepare all protocol-required tissues for all dose groups to slides (H & E) and perform histopathological examination on tissues from the high dose and control groups. If treatment-related findings are seen in high dose animals for a reproductive tissue, then reproductive tissues for the effected gender at all dose levels shall be examined. For costing purposes, assume that microscopic treatment-related findings are seen in testes and epididymis from high dose animals, triggering the evaluation of male reproductive tissues at all dose levels.

F1c mating and F2 assessment:

At approximately 13 weeks postnatal, randomly assign 2 males and 2 females from each F1c litter to create up to 50 non-sibling mating pairs per dose group, for the

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purpose of producing three (3) F2 litters (F2a, F2b, F2c) for assessment. (For costing purposes assume 50 pairs per group).

The production and assessment of F2 litters shall be the same as the F1 litter production and assessment. Once the F2c litter is produced, continue treatment of these offspring until study termination. Study termination shall generally be weaning (PND 21) or, if effects were seen in the prior generation that will be further investigated, treatment will continue until sexual maturation (approximately 13 weeks).

For costing purposes, assume that the F2c’s shall be maintained until 13 weeks of age.

For F2c animals carried through weaning to adulthood, assess the F2c males for the presence of nipples at PND 13, testicular descent, prepuce separation, and the presence of nipples on shaved abdomens of adults at necropsy (if nipples were seen at PND 13); and the F2c females for the day of vaginal opening. Vaginal cytology data on F2c females shall be collected 16 days prior to euthanasia.

The Contractor shall submit a draft summary on litter size and sex distribution and weight for each litter (i.e., F2a, F2b and F2c) within 5 days after the last expected delivery for that littering. After birth of the last F2c litter, the Contractor shall submit a draft summary of littering data and survival to the COR within 5 days. Pup survival data shall be submitted to the COR within 5 days of the last PND21 for each littering.

F1c Breeder Necropsy:

Necropsy F1c animals after all of their F2c offspring have been weaned and vaginal cytology assessment of F1c females is complete. At necropsy of F1c animals, collect the same sperm measurements, weights, and tissues that were specified in the F0 necropsy and process all tissues to slides. In addition, remove and fix nipples if found in the F1c male offspring. Slides for high dose and control animals shall be read. If abnormal findings are seen for a tissue, all dose groups for that tissue shall be read. For costing purposes, assume that microscopic treatment-related findings are seen in testes and epididymides from high dose animals, triggering the reading/evaluation of male reproductive tissues at all dose levels. In addition, assume retained nipples are seen in 10 F1c high dose males that must be collected, processed and evaluated.

F2c Necropsy:

After measurements of sexual maturation have been completed for the F2c animals, necropsy shall be performed (for this RFP, assume at 13 weeks of age). Collect organ weights and tissues as for the F1c animals and process all protocol-required tissues to slides for all dose groups.

Histopathologic evaluation shall be performed on tissues in the high dose and control groups. For costing purposes, assume that microscopic treatment-related findings are seen in testes and epididymis from high dose animals, triggering the evaluation of male reproductive tissues at all dose levels. In addition, 13 ATTACHMENT 6 assume that retained nipples are seen for 10 high dose males that must be collected, processed and evaluated.

11) Final Report

Within 26 weeks of the final euthansia, an audited draft final report including materials and methods, results, data tables, discussion, summary and references shall be delivered to the NTP. The report shall describe all pertinent information, including data on fertility of breeding pairs and number of pups per litter for each breeding segment of the study.

The draft final report shall be audited and considered, in the contractor’s estimation, a final report subject only to review and approval by the COR. If a review of the draft final report results in changes, a new version of the draft final report reflecting the required changes must be submitted.

Upon approval of the draft, the final report shall be sent to the NTP as a hard copy and in electronic format (PDF) on a CD-R CD. All final reports shall be dated and signed by the individual(s) responsible and the QAU. Reports are to be saved or scanned at 300 dpi (black and white) to a single searchable PDF file. The file should have no password protection and no document property restrictions. Reporting requirements are listed in the table at the end of the primary SOW for this contract.

All reports shall be sent to the appropriate recipient as indicated in the table. The COR will supply names of individual recipients as indicated.

12) Protocol Changes

The COR shall be consulted and agree in advance to any changes in the protocol.

The written protocol amendment and justification shall be signed by both the contract lab Study Director and the COR, and maintained with the original approved protocol.

13) Performance/Delivery Requirements

See main SOW and use instructions for studies over one year in duration. All original source documentation/data for the study shall be sent to the NTP Archives, including all paper records, electronic records, tissues, slides, blocks etc. Archival materials shall be appropriately boxed and shipped with an Inventory/Index organization scheme that has been approved by the COR.