MOG_Sample_Study_Final.pdf

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SAMPLE STUDY ASSIGNMENT #3

STUDY TITLE: PROTOCOL OUTLINE FOR THE NTP MODIFIED ONE GENERATION

STUDY (MOG) OF DIMORPHOLINODIETHYL ETHER (CAS # 6425-39-4) IN

HARLAN SPRAGUE DAWLEY RATS ADMINISTERED DAILY BY GAVAGE

1. INTRODUCTION

The test material for this study is dimorpholinodiethyl ether. It is a blowing agent for the production of flexible, molded, and moisture-cured foams and coatings. It was nominated to the NTP testing program due to a paucity of information on its toxicity.

2. OBJECTIVE

To characterize the reproductive and developmental toxicity of dimorpholinodiethyl ether in the Harlan Sprague Dawley rat using the NTP’s modified one-generation (MOG) study design. Offspring in this study shall be evaluated for a variety of endpoints.

The MOG is composed of two interrelated parts: (1) a dose range-finding (DRF) study conducted with dosing from Gestation Day (GD) 6 through weaning to determine the maximum dose that is tolerated by the dam and has no impact on perinatal survival until weaning; and, (2) a definitive MOG where time-mated females are administered the test material from GD 6 through weaning, and the subsequent F1 offspring are then continuously administered the test article via the same route of exposure as the dams. F1 animals retained after PND 4 are taken to adulthood for gross and histo-pathology examination and can be allocated to various cohorts for evaluations such as fertility/fecundity, prenatal developmental toxicity (teratology), 13-week subchronic toxicity, immunotoxicity, biological sampling, maternal transfer (of test article), neurotoxicity, or others, depending on the nature of the test article and the evaluations required. For this study, extra animals shall be ordered for collection of samples to evaluate maternal transfer of test material (sample evaluation shall be conducted at an NTP-specified laboratory).

In actual practice, only certain cohorts will be selected for each MOG study depending on the test article, the needs of the program and the number of offspring per litter. However, for the purpose of this RFP, the offer shall provide a technical approach and a cost for the following: Prestart Chemistry, Range-finding study, and Definitive Study. For the definitive study, a break down of cost for each individual cohort is needed, as well as a separate cost for the F0 Gestation and Lactation phase.

Therefore, costs for the following are needed:

Prestart Chemistry, Range-finding study, Full study F0 Gestation and Lactation phase, Fertility/fecundity cohort, Teratology cohort, Neurotoxicity cohort, Immunotoxicology cohort, Biological Sampling cohort, 13 week Subchronic toxicity cohort, 1 Year toxicity cohort, Maternal Transfer.

The contractor shall provide an estimate of the amount of test article required for this study.

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3. TEST MATERIAL

3.1. NAME

Dimorpholinodiethyl ether

3.2. CAS #

6425-39-4

3.3. CHARACTERIZATION

Bulk chemical will be characterized by NTP before shipment to the toxicology testing lab. After arrival at the toxicology testing lab, bulk chemical analysis to confirm identity and purity shall be performed (using methods provided by NTP); then bulk chemical reanalysis shall be performed every 24 +/- 2 weeks until within 30 days of the end of the definitive study,

3.4. PRE-START CHEMISTRY ACTIVITIES: TO BE PERFORMED PRIOR TO DOSING ANIMALS

Receive the test material and conduct bulk chemical analysis (identity and purity confirmation) according to the procedures provided by NTP; 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 as outlined in the NTP Specifications;

Prepare a Pre-Start Chemistry Report. This report must be received and accepted by the COTR before animals are dosed. The pre-start chemistry report shall include:

o A summary of the NTP-provided information on the source of the bulk chemical, storage requirements, identity and purity data;

o Confirmatory data on identity and purity (including representative chromatograms and spectra of the test article and reference material);

o provide copies of the SOPs for the bulk chemical reanalysis methods;

o Data on preparation, handling, homogeneity, and analysis of dose formulations as well as formulation SOPs;

o Dose formulation analysis method performance evaluation procedures and validation data, including representative chromatograms or spectra;

o Health and safety procedures to be followed when using dimorpholinodiethyl ether.

3.5. ROUTINE FORMULATION ANALYSES

50 mL representative samples from each dose level shall be analyzed for concentration determination from the first set of formulations prepared for the study and from the animal room on the last dosing day using this formulation set. The schedule for routine analysis of formulations and animal room samples is dependent on the length of the study, formulation stability and other factors. For costing purposes, assume formulation checks shall be conducted approximately every 10 weeks in the definitive study and at the beginning and end of the range-finding study.

3.6. ROUTE OF ADMINISTRATION

Gavage (dose volume 5 mL/kg)

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3.7. VEHICLE INFORMATION

Deionized water

4. TEST ANIMALS

4.1. SPECIES, STRAIN/STOCK AND AGE

Time-mated (assumed pregnant) female Harlan Sprague Dawley Rats (Harlan Laboratories). F0 female rats shall be sexually mature (11-12 weeks; 200-225 g) and mated at the supplier. F0 female rats shall be obtained on or before gestation day (GD) 2. Day of positive evidence of mating = GD 0.

F1 male and female rats shall be sexually mature when mated.

4.2. DIET, WATER, BEDDING AND ENVIRONMENT

Tap water and NIH-07 feed provided ad libitum (feed shall be obtained from an NTP-approved vendor, Ziegler Bros., Gardners, PA). For cohorts that will not have a breeding component, animals shall be provided NTP-2000 after weaning. Feed shall be stored at 70F or lower and 50% humidity or lower, and in a well-ventilated area stored raised above the floor. Batch numbers for feed and use periods shall be recorded. Feed shall be used for no more than 120 days post milling. Laboratories must demonstrate that drinking water provided for animals meets US/EPA National Primary Drinking Water Regulations (analysis at least once/year).

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.

The light cycle in the animal rooms shall be 12-hour light/dark. The temperature of the animal rooms shall be maintained at 72 +/- 3F. 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.

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 the lactation period, dams shall be housed with their litters.

4.3. PRE-STUDY EVALUATION AND SENTINELS

Animals shall be examined by the veterinarian before being released for study. Non-pregnant animals from the vendor’s same animal room shall be purchased to be used for pre-study health evaluation and sentinel controls for the study. Ten of these non-pregnant females shall be used for the pre-study evaluation for internal and external parasites, and serum shall be collected for serology screening to be performed at an NTP-specified laboratory (Radil, Univ. of MO, 1600 E. Rollins, Columbia, MO 65211) for organisms outlined in the NTP Specifications for Reproductive Studies. Sentinels are required for studies over 6 weeks in duration. Time points for sentinel and disease screening are: pre-study, 4 weeks later and at least every 6 months. While the pre-

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study and 4 week animals are purchased from the supplier, culls shall be used for the sentinels examined at 6 month intervals. At 6 month intervals during the course of the study, sentinels (5/sex) shall be bled only (not necropsied). At the study termination, the sentinels shall be bled and necropsied. For sentinel/disease screening animals, body weights and food consumption shall not be measured during the study. If cohorts of animals are moved to a separate animal room, appropriate sentinels must be included for these animals.

4.4. RANDOMIZATION AND IDENTIFICATION

On GD3, F0 female rats shall be randomized into treatment groups using a method to minimize the variance in mean body weight, and uniquely identified by tail tattoo. F1 pups shall be identified by paw tattoo according to number within litter at PND 1 and uniquely identified at weaning by tail tattoo.

4.5. CAGING

Dams shall be housed one per cage (except when with their pups). Pups shall remain with their respective dam until weaning on PND 28 (or euthanasia). After weaning, juveniles shall be group housed by litter and sex (up to 5 per cage as appropriate for their size). When in co-habitation for mating, rats shall be housed as pairs of one male and one female (non-siblings) per cage from the same dose group. After evidence of mating, or after 15 days if no evidence of mating, the pairs shall be separated and individually housed until necropsy.

4.6. EUTHANASIA

Discomfort or injury to animals shall be limited, in that if any animal, including pre-weaning Fl or F2 offspring, becomes severely debilitated or moribund (as well as all rats at scheduled necropsy/termination) it shall be humanely euthanized according to the laboratory’s IACUC approved procedures acceptable to the COR.

5. DOSE RANGE-FINDING STUDY (DRF): STUDY DESIGN

5.1. DOSE LEVELS AND NUMBER OF RATS

Rats shall be randomized as described in Section 4.4 into 6 groups and administered the test material daily via gavage as indicated below:

Group Dose (mg/kg)

No. of F0 females

Control (1) 0 14

2 50 14

3 100 8

4 250 8

5 500 14

6 1000 14

Total 72

5.2. DURATION OF DOSING

Females shall be dosed daily from GD 6 through postnatal day (PND) 28. PND 0 is defined as the day that all pups within a litter have been delivered. Direct

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dosing of pups shall begin on PND 12. Dose volume shall be based on the most recent dam and pup body weight.

5.3. IN-LIFE ENDPOINTS

Out of cage clinical observations once daily (1-3 hrs post dose); cage side observations twice daily (before 10AM and after 2PM) including signs of delivery, moribundity and/or death. Care shall be taken so that dams in the process of giving birth are not disturbed. Pup observations daily.

Dam body weights recorded daily during gestation and lactation (day of randomization onwards). All weights are to be reported as well as gestational/post-natal weight gains for the following intervals: GD 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, and 6-21; PND 1-4, 4-7, 7-10, 10-14, 14-17, 17- 21, 21-24, 24-28, PND 1-21, and PND 1-28.

Dam feed consumption measured for GD 3-6, 6-9, 9-12, 12-15, 15-18, 18- 21, and PND 1-4, 4-7, 7-14, 14-21, 21-28 (report for these intervals as well as GD 6-21 and PND 1-21 and PND 1-28).

Number of live and dead pups delivered by sex and by litter and total on

PND 0.

Number and weight of live pups by sex and by litter and total on PND 1, 4, 7, 14, 21, 28. Litters shall not be culled in the DRF.

Number of pregnant and non-pregnant rats; duration of pregnancy.

5.4. BIOLOGICAL SAMPLE COLLECTION

On GD 18, confirmed pregnant dams (3 dams/dose group at 0, 50, 500 and 1000 mg/kg) shall be euthanized and the following collected:

Maternal plasma isolated from trunk blood (at least 3.0 ml [target]).

Amniotic Fluid (pooled by litter; at least 250 ul [target]; stored at -70 +/- 3oC)

Fetuses (pooled by litter, frozen in liquid nitrogen)

On PND 4, dams and their pups (3 dams/group at 0, 50, 500 and 1,000 mg/kg) shall be euthanized and the following collected:

Maternal Plasma (at least 3.0 ml [target])

Whole pup in a manner appropriate for chemical analysis (3 per sex/litter)

Samples shall be labeled and stored individually in a manner so that it is clear which pups belong to which dam.

Fetuses/pups and plasma shall be sent to the designated NTP Chemistry contractor for analyses (address provided by COR when needed).

Frozen samples shall be stored at a temperature of -70 +/- 3oC or lower.

NOTE- If the number of apparently pregnant dams is insufficient to obtain biological samples as well as range-finding information, the COR shall be contacted prior to biological sample collection for direction.

5.5. EUTHANASIA

Rats shall be euthanized by Contractor-IACUC-approved procedures acceptable to the NTP COR. Females and pups shall be euthanized on PND 21 (or sufficiently after expected littering date, if no evidence of pregnancy).

Rats that produced litters, and pups shall be discarded without examination.

Rats that do not appear to be pregnant and do not deliver shall be examined

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for the presence of uterine implantation sites (using ammonium sulfide, Prussian blue, or other appropriate technique) and corpora lutea enumerated.

5.6. REPORTING

Data summaries shall be provided to the COR within five days of delivery of the last litter and within 5 days of the euthanasia of the last DRF animal on study.

Data shall be analyzed using standardized parametric and non-parametric tests available within the computerized data management system.

Mean values and index of variability (i.e. SE, or SD) for all protocol specified parameters shall be provided, including (but not limited to):

• Number pregnant, survival, clinical observations, mean body weights, body weight gains, food consumption

• Dams that do not deliver- number of resorptions, implants, calculated pre and post implantation loss.

• Mean litter size, mean body weight of live pups (by sex, and combined) and total number live/dead pups.

• Pup survival on PND 1-4, 4-7, 7-14, 14-21, 21-28; and 4-21 and 4-28.

6. DEFINITIVE STUDY: STUDY DESIGN

6.1. DOSE LEVELS AND NUMBER OF ANIMALS

Time-mated rats shall be randomized by body weight into 4 groups:

Group Dose levelsa

(mg/kg) No. of F0 time mated femalesb

Vehicle Control 0 30

Low Dose 100 30

Mid-Dose 500 30

High-Dose 1000 30

Total F0 120 a: F1 offspring shall be administered the test article starting on PND 12 and continuing post-weaning at the same dose as their parents b: aiming to have at least 23 litters/dose group.

6.2. DURATION OF DOSING

F0 dams shall be treated by gavage daily from GD 6 throughout gestation, lactation and continuing until necropsy. (For technical and business proposals, assume that F0 dams are euthanized at weaning.)

F1 and F2 generation pups shall be administered the same dose as their dam and dosing of pups shall begin on PND 12. Dosing shall continue 7 days a week until euthanasia. Dose volume administered shall be based on the most recent body weight for dams and pups.

6.3. F1 PUP IDENTIFICATION, LITTER STANDARDIZATION AND USE OF CULLS

F1 pups shall be uniquely identified on PND 1 by paw tattoo. F1 litters shall be standardized to 10 pups (as close to 5 male and 5 females as possible) on

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PND 4 if litter size is larger than 10 pups. Contractor shall communicate with the COR regarding the number of litters and pups available for study.

Inguinal mammary glands from ten males/dose level and ten females/dose level randomly selected from PND 4 F1 culls (from different litters) shall be collected and whole mounted for subsequent assessment of abnormal mammary gland development.

Non-selected F1 pups shall be euthanized on PND 4. Note- if it is not possible to determine sex on PND 4 (i.e. apparent skewed sex ratio), litter standardization may need to be delayed until weaning. FOR COSTING

PURPOSES ASSUME THIS WILL BE DONE AT PND 4.

6.4. IN-LIFE ENDPOINTS

6.4.1 F0 dams

Out of cage clinical observations once daily; cage-side observations twice daily (before 10AM and after 2PM) including signs of delivery, moribundity and/or death. Care shall be taken so that dams in the process of giving birth are not disturbed.

Female body weights daily during gestation and lactation (day of randomization onwards) and at termination. Body weight changes to be reported for the following intervals: GD 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, PND 1-4, 4-7, 7-10, 10-13, 13-16, 16-19, 19-

21, 21-24, 24-28; PND 1-16; 1-21, 1-28, 4-21, 4-28;

Feed consumption: GD 3-6, 6-9, 9-12, 12-15, 15-18, 18-21; PND 1-4, 4-7, 7-10, 10-13, 13-16, 16-19, 19-21, 21-24, 24-28; and

PND 1-16; 1-21, 4-21, 4-28.

Number of pregnant females and duration of pregnancy.

6.4.2 F1 offspring: Lactation Period

PND 0: Number of live and dead offspring (by litter, sex, and total)

PND 1: Number of live and dead offspring (by litter, sex, and total); anogenital distance (AGD) and corresponding pup weight.

PND 4: Number of live and dead pups, sex, pre-cull weight (by litter, sex, and total).

Presence and type of developmental anomalies in the pups (e.g.

clinical observations, gross external malformations).

Number and weight of pups on PND 4 (post cull), 7, 10, 13, 16, 19, 21 and 28; (by pup, sex and litter). Pup body weight gain changes by sex to be reported for the following intervals 1-4 pre cull, 4 (post cull)-7, 7-10, 10-13, 13-16, 16-19, 19-21, post cull 4- 28.

Males: Presence of areolae on PND 13, testicular descent (PND 14-30) and body weight on day of acquisition for testicular descent.

6.5. F0 NECROPSY

F0 female rats shall be necropsied after weaning has been completed. Animals shall be subjected to an external examination and uterine implantation scars enumerated. Gross lesions and representative control tissues shall be

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retained, processed to slides and examined histopathologically. (For Costing Purposes Assume 5 female animals in the high dose group each have one gross lesion that is collected and examined.) Rats that do not appear to be pregnant and do not deliver shall be examined for the presence or evidence of uterine implantation sites (using ammonium sulfide, Prussian blue, or other appropriate technique) and corpora lutea enumerated. (For Costing Purposes, Assume 2 F0 female rats out of 30 per dose group do not litter.

Assume the other 22 dams and litters per dose group shall be kept on study until weaning.)

6.6. F1 PUP ALLOCATION TO COHORTS

Pups shall be randomly allocated to cohorts at weaning. NOTE: Cohorts to be studied will be test-article specific and may include one or more of the cohorts indicated in the table below. It is anticipated that a “typical study” will include 3 cohorts, such as fertility, teratology and 13-week subchronic toxicity. However, this will vary from one test article to another. Note: This sample study assignment is provided for offerors to prepare technical and business proposals only. Each cohort should be costed separately. Cost the “F0 gestation and lactation” phase separately assuming that 28 out of 30 litters have 12 pups each and are standardized to 5 male and 5 female pups. In the technical proposal, include information to address procedures if not all litters have 12 pups.

Cohorts Number of male and female pups/litter

Total number of male pups/dose

Total number of female pups/dose

Total number of pups/cohort

Fertility/fecundity 1 male and 1 female 20 20 160

Prenatal Toxicology (Teratology)

1 male and 1 female

23 23 184

13 Week Subchronic 1 male and 1 female 20 20 160

Neurotoxicology 1 male and 1 female 20 20 160

Post weaning Mammary Gland/ Biological Samples

1 male and 1 female

20 20 160

Immunotoxicity 1 male and 1 female 20 20 160

1 Year Study 1 male and 1 female 25 25 200

Note- in an actual study, only selected cohorts would be run and the number of animals in each cohort may be slightly different. For the maternal transfer study, a separate group of 12 pregnant females is required.

Approximate length of time to conduct each cohort (after weaning) is given in the table below. The F0 phase for gestation and lactation is approximately 49 days (dam arrival ~GD2, with dosing GD 6 to PND 28).

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Phase of study Days Post-Weaning

F1 Fertility (Breeding) cohort up to 136 days;

up to 164 w/crossover

F1 Prenatal Tox (Teratology) cohort up to 130 days

F1 Neurotoxicity cohort ~91 days

F1 Immunology cohort ~4 days

Biological Sample/ Post Weaning Mammary Gland cohort

Varies depending on study design

F1 13 Week Subchronic cohort ~91 days

F1 1 Year cohort ~52 weeks

6.7. NECROPSY OF NON-SELECTED F1 OFFSPRING

Non-selected F1 offspring shall be euthanized and given a gross necropsy.

(For costing, assume that there are 20 non-selected pups/sex/dose group). Note: it is recognized that the number of activities required in this sample study assignment uses more pups than are available from 30 dams per group described here.

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THE FOLLOWING SECTIONS DESCRIBE MULTIPLE COHORTS of which generally 2-4 of these would be selected for an actual study. The offeror must provide a separate cost for each cohort. For estimating the cost of a typical study for determining costs per year, assume a typical MOG will have the fertility, teratology and 13-week cohorts.

7. PRENATAL TOXICOLOGY (TERATOLOGY) COHORT

7.1. ALLOCATION AND COHORT OVERVIEW

F1 offspring shall be randomly allocated to the prenatal toxicity cohort as described in Section 6.6. Vaginal cytology shall be initiated at 77 ± 3 days of age (prior to cohabitation). After 16 days of vaginal cytology (week 13), males and females from the same dose group shall be paired 1:1 to a non-sibling for cohabitation. After evidence of mating (or after 15 days if no evidence of mating), males and females shall be separated and females shall be terminated on GD21 for fetal evaluations (dosing not required on day of necropsy). Males shall be necropsied after cohabitation and no forther evaluations are required.

7.2. IN-LIFE ENDPOINTS

Out of cage clinical observations once daily; cage-side observations twice daily (before 10AM and after 2PM) including signs of moribundity and/or death;

weekly body weights and cage feed consumption for males and for females until evidence of mating

Males: balano-preputial separation (BPS) PND 35 until acquisition (the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition.

Females: day of vaginal opening (VO) PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of acquisition; estrous cyclicity (starting at PND 77 ± 3 days and continuing for 16 days).

F1 Dams (upon evidence of mating)

Time to mating (pre-coital interval)

Out of cage clinical observations once daily; cage-side observations twice daily (before 10AM and after 2PM) including signs of delivery, moribundity and/or death.

Female body weights daily during gestation. Body weight changes to be reported for the following intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21, 6-21

Feed consumption; GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21, 6-21

Number of pregnant females

7.3. F1 DAM NECROPSY AND FETAL EVALUATION

The following endpoints shall be measured:

Maternal body, liver, adrenal (paired), ovarian (left and right separately), and gravid uterine weights.

Pregnancy status and the number of corpora lutea. In the gravid uterus, the numbers of deaths (resorptions (early vs. late) and/or dead fetuses) and live fetuses shall be recorded. Rats that do not appear to be pregnant shall be

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examined for the presence or evidence of implantation sites (using Prussian Blue or other appropriate method).

For each dam, the position of each fetus (or implant) in the uterine horns, starting at the left end, noting the cervix and moving to the right end, shall be noted. All live fetuses shall be counted, weighed, and sexed (visual anogential distance). The placentas from live fetuses shall undergo gross observation.

All fetuses (and late resorptions to the extent possible) shall be examined for external morphological abnormalities, including cleft palate.

Fetuses shall be euthanized, internally sexed and examined for visceral morphological abnormalities via Staple’s technique. Approximately one-half of the same fetal carcasses shall be decapitated prior to dissection.

Fetal heads shall be fixed and decalcified in Bouin’s solution, and subsequently examined for soft tissue craniofacial alterations.

All fetal carcasses shall be eviscerated, and the skeletons macerated and stained with Alcian Blue/Alizarin Red S stain. All fetal skeletons shall be examined for skeletal morphological abnormalities.

7.4. F1 MALE NECROPSY

Males shall be necropsied after the cohabitation period and shall be subjected to a gross examination.

The following endpoints shall also be measured:

Left cauda epididymal spermatzoa measurements: number, density, motility.

Left testicular measurements: spermatid head count. Note- in the case of unilateral effects on organ weights, or presence of gross lesions, pathology takes precedence over sperm assessments in selection of right or left testis/ epididymis.

Organ weights:

Adrenal glands (paired) Dorso-lateral Prostate

Epididymis, right and left Ventral Prostate

Cauda Epididymis, right and left Preputial glandsa

Testis, right and left

Seminal Vesicles (w/ Coagulating Glands)

Levator ani bulbocavernosusa a: Triggered tissue: Only collected and weighed when an antiandrogenic effect is expected (e.g.

observed increased incidence of areolae, delay in BPS). For this RFP, assume these will be weighed.

7.5. REPORTING

See Section 15 and the NTP Specifications for Reproductive Studies.

8. FERTILITY COHORT

8.1. ALLOCATION AND COHORT OVERVIEW

F1 offspring shall be randomly allocated to the Fertility cohort as described in Section

6.6. Vaginal cytology shall be initiated on PND77+ 3 (prior to mating) and smears taken for 16 days. After 16 days, males and females from the same dose group shall be paired 1:1 to a non-sibling for cohabitation. After evidence of mating (or after 15 days if no evidence of mating), males and females shall be separated and females

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shall be allowed to litter. F2 pups shall remain with their dams until weaning. F2 pups shall be evaluated for attainment of developmental landmarks. Male F2 offspring shall be euthanized on PND 50 and female F2 offspring shall be euthanized after vaginal cytology is complete.

8.2. IN-LIFE ENDPOINTS

(before 10AM and after 2PM) including signs of moribundity and/or death; Care shall be taken so that dams in the process of giving birth are not disturbed.

Weekly body weights and cage feed consumption for males and for females until cohabitation.

Males: balano-preputial separation (BPS) PND 35 until acquisition (the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition.

Females: day of vaginal opening (VO) PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of

F1 dams

Time to mating (pre-coital interval)

Female body weights daily during gestation and PND 1, 4, 7, 10, 13, 16, 19 and 21; at termination. Body weight changes to be reported for the following intervals:

GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21, PND 1-4, 4-7, 7-10, 10-13, 13-

16, 16-19, 19-21; PND 1-16, 1-21; 4-21

Feed consumption twice weekly (when not mated/pregnant); GD 0-3, 3-6, 6-9, 9- 12, 12-15, 15-18, 18-21 PND 1-4, 4-7 7-10, 10-13, 13-16, 16-19, 19-21 and PND 1- 16; 1-21; 4-21 (NOTE- twice weekly is defined as every three, then 4 days)

Number of pregnant females and duration of pregnancy.

F2 offspring

PND 0: Number of live and dead offspring (by litter, sex, and total)

PND 1: Number of live and dead offspring (by litter, sex, and total); anogenital distance (AGD) and corresponding pup weight.

PND 4: Number of live and dead pups, sex, pre-cull weight (by litter, sex, and total). Pups will be culled to 4 males and 4 females/litter when possible.

Presence and type of developmental anomalies in the pups (e.g. clinical observations, gross external malformations).

Number and weight of pups on PND 4 (post cull), 7, 10, 13, 16, 19, 21 and 28 (by pup, sex and litter). Pup body weight gain changes by sex to be reported for the following intervals 1-4 pre cull, 4 (post cull)-7, 7-10, 10-13, 13-16, 16-19, 19-21, 21- 28, post cull 4-21.

Males: Presence of areolae on PND 13, evaluation of testicular descent (PND 14- 30), balano-preputial separation (PND 35 until acquisition; the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition shall be collected.

Females: evaluation of day of vaginal opening PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of acquisition.

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8.3. F1 PARENTAL NECROPSY

Females shall be necropsied after the F2 offspring have been weaned.

Males shall be necropsied after potential effects on fertility have been ascertained (see Section 8.5 on Determination of Affected Sex). If areolae are noted on male rats at PND 13, the ventral surface of male rats shall be shaved and nipples counted at necropsy. Assume 5 male high dose rats have nipples at PND 13.

Trunk blood shall be collected and plasma isolated (target volume 2mL), frozen and shipped to an NTP designated lab for hormone (or other) analyses as directed by the COR.

The following endpoints shall also be measured:

Left cauda epididymal spermatzoa measurements: number, density, motility.

Left testicular measurements: spermatid head count. Note- in the case of unilateral effects on organ weights, or presence of gross lesions, pathology takes precedence over sperm assessments in selection of right or left testis/ epididymis.

Organ weights:

Adrenal glands (paired) Dorso-lateral Prostate

Epididymis, right and left Ventral Prostate

Cauda Epididymis, right and left Ovaries, right and left

Testis, right and left Thyroid Gland

Seminal Vesicles (w/ Coagulating Glands) Preputial glanda

Levator ani bulbocavernosusa a: Only collected and weighed when an antiandrogenic effect is expected (e.g. decrease in AGD, observed increased incidence of areolae, or a delay in BPS). For this RFP, assumed they are weighed.

Fix and process the following tissuesa per NTP specifications:

Adrenal Glands Ventral Prostate

Vagina, cervix, and uterus (together- as per NTP direction)

Ovariesc

Epididymis, right b Pituitary

Testis, rightb Thyroid Gland

Seminal Vesicles (w/ Coagulating Glands) Gross lesions

Dorsal Prostate Nipple/adjacent mammary glandd a: All tissues shall be fixed in neutral buffered formalin with the exception of the testes and epididymides which shall be fixed in modified Davidsons b: If the rat exhibits a gross findings on the left, rather than the right, collect, process, and examine the left tissues as well for histopathology c: Step sectioned and primordial follicles enumerated d: One retained nipple from each male with retained nipples shall be collected for histopathologic evaluation. (Assume 5 male rats in the high dose group have retained nipples.)

Fixed tissues shall be processed to slides and stained with hemotoxylin and eosin for all dose groups. The high dose and control groups shall be examined for histopathological changes. If high dose level effects are found, all the remaining dose groups shall be examined for these and any related changes as defined by the COR.

14 ATTACHMENT 6

8.4. F2 NECROPSY

Pups shall be necropsied on PND 50 for males and after vaginal cytology is complete for females. These animals shall be given an external and internal examination. All gross lesions and comparative controls shall be retained and processed for histopathological evaluation. (For costing, assume that 5 high dose males and 5 high dose females each have one gross lesion (testicular or uterine) that is collected along with a respective control.)

8.5. DETERMINATION OF AFFECTED SEX – CROSS-OVER MATING

(For this RFP, assume a cross-over mating shall be conducted.) If a decrease in fertility and/ or fecundity has been attributed to test article exposure (e.g. a decrease of 2 or more pups per litter), or as otherwise requested by the COR, the Contractor shall conduct a cross-over mating of the F1 animals. A cross-over mating shall consist of (1) mating all the high dose males with test article naive nulliparous females (i.e. newly purchased); and (2) mating all high dose females with control males. Exposure to test article shall be suspended for the duration of mating. Test article naïve nulliparous females shall be purchased in a number sufficient for there to be one-to-one mating pairs. Test article naïve nulliparous females shall be sexually mature (11 weeks of age) when mated with the high dose males. Mating pairs of 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. Litters from the cross-over mating shall be evaluated through PND 4. The following data shall be collected: number of females plugged/sperm positive, 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 and corpora lutea counted. The high dose and control males shall be terminated if it is determined by the COR that no further testing of them (such as dominant lethal test) is required.

8.6. REPORTING

See Section 15 and the NTP Specifications for Repro Studies.

9. NEUROBEHAVIORAL COHORT

9.1. ALLOCATION

F1 offspring shall be randomly allocated to the Neurobehavioral Toxicity cohort as described in Section 6.6.

9.2. IN-LIFE ENDPOINTS

(before 10AM and after 2PM) including signs of, moribundity and/or death;

weekly body weights and cage feed consumption for males and for females until evidence of mating

Males: balano-preputial separation (BPS) PND 35 until acquisition (the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition.

Females: day of vaginal opening PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of

15 ATTACHMENT 6

9.3. MOTOR ACTIVITY

Motor activity shall be monitored by an automated activity recording apparatus on PND 13, 17, 21, and 60 (+2 days) (N=10/sex/group).

9.4. AUDITORY STARTLE TEST

An auditory startle habituation test shall be performed on the offspring around the time of weaning and approximately PND 60 (N=10/sex/group).

9.5. LEARNING AND MEMORY TEST

A test of associative learning and memory shall be conducted around the time of weaning and aproximately PND 60 (N=10/sex/group).

9.6. NEUROPATHOLOGY.

Neuropathological evaluation shall be conducted on animals on postnatal day 11 and at the termination of the cohort. At 11 days of age, ten male and ten female pups per dose group shall be euthanized for neuropathological analysis. The pups shall be killed by exposure to carbon dioxide and immediately thereafter the brains removed, weighed, and immersion-fixed in an appropriate aldehyde fixative. At the termination of the cohort, one male or one female from each litter (10/sex/dose) shall be killed by exposure to carbon dioxide and immediately thereafter the brain shall be removed, weighed and immersion-fixed for neuropathological evaluation. Neuropathological evaluation shall be conducted on 10/sex/dose for all dose groups.

9.7. REPORTING

10. IMMUNOTOXICOLOGY COHORT

F1 animals shall be allocated as outlined in Section 6.6 and transferred on PND 25 to an NTP designated contractor with sufficient amount of feed to last until PND 50. Arrangements must also be made to supply dose formulations to last until PND 50. (The Contractor performing the MOG study is expected to produce and supply dosing formulations to the immunotoxicology laboratory.) Study scheduling and animal shipment must be coordinated with the NTP Immunology Discipline leader and the NTP designated contractor prior to mating the F0 females. Contact information (including phone and email) for the individual coordinating the shipment, and serology studies attesting to the health status of sentinel rats housed in the same room as the study animals must be received at the NTP designated Immunotoxicology contract laboratory a minimum of 21 days prior to shipment of the F1 Immunotoxicology cohort.

11. (POST) WEANING MAMMARY GLAND/ BIOLOGICAL SAMPLING COHORT

11.1. ALLOCATION

F1 offspring shall be randomly allocated to the post weaning mammary gland;

biological sampling cohort as described in Section 6.6.

16 ATTACHMENT 6

11.2. IN-LIFE ENDPOINTS

(before 10AM and after 2PM) including signs of moribundity and/or death;

weekly body weights and cage feed consumption for males and for females.

11.3. POST WEANING MAMMARY GLAND ANALYSES

The inguinal mammary glands (4th and 5th) from one F1 offspring/sex/litter at the time of vaginal opening (female) or PND 21 (male) shall be collected and whole mounts prepared (ten/sex/dose level at the appropriate time). These animals shall be given a gross examination and tissues with gross lesions (and comparative controls) shall be fixed and retained for subsequent histopathological evaluation. For costing, assume there are no gross lesions.

11.4. BIOLOGICAL SAMPLING

On PND 21 (F1 male) and at vaginal opening (F1 female), 5 animals/sex from each dose group used for mammary gland analysis in 11.3 shall have the following collected:

Plasma isolated from trunk blood (at least 1.5 ml [target]), kidneys (paired), epididymides (paired), testes (paired), ovaries (paired), and liver (left lateral lobe;

~1g); flash frozen.

Plasma and tissues shall be sent to the designated NTP Chemistry contractor (address provided by COR when needed).

Frozen samples shall be stored at a temperature of -70 ± 3oC or lower.

12. SUBCHRONIC (13 WEEK) TOXICITY COHORT

12.1. ALLOCATION

F1 offspring shall be randomly allocated to the Subchronic Toxicology cohort as described in Section 6.6 along with appropriate sentinel animals if needed. There shall be 10/sex/dose group designated as Core animals and 10/sex/dose group designated as Clinical Laboratory Study animals.

12.2. DIET

For animals in the subchronic cohort, the diet shall be NTP-2000 starting after weaning.

12.3. IN-LIFE ENDPOINTS

Out of cage clinical observations once weekly; cage-side observations twice daily (before 10AM and after 2PM) including signs of, moribundity and/or death;

weekly body weights and cage feed consumption balano-preputial separation (BPS) PND 35 until acquisition (the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition.

Females: day of vaginal opening (VO) PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of

17 ATTACHMENT 6

12.4. SUBCHRONIC (13 WEEK) TOXICOLOGICAL PARAMETERS TO BE EVALUATED (RESULTS MAY

BE USED TO AID IN SELECTION OF DOSES FOR SUBSEQUENT STUDY)

12.4.1 F1 Clinical Laboratory Studies

Ten of the predesignated rats shall be used for collections at days PND 24 ± 2 and PND 42 ± 4 for clinical laboratory studies. Blood shall be collected from the retro-orbital sinus under CO2/O2 anesthesia at days 24 ± 2 & 42 ± 4 from the satellite rats and these animals shall be sacrificed after the collection on PND 42. Inguinal mammary glands shall be taken from these males and females for whole mount evaluation as per the NTP Specifications for Repro Studies.

The remaining Core study rats shall be bled at terminal euthanasia for hematology determinations and for clinical laboratory studies.

All male and female animals shall be treated for approximately the same number of days before collection of samples, and all animals of a sex shall be bled on the same day where possible.

Hematology Erythrocyte count Hemoglobin concentration Hematocrit (Packed cell volume) Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Leukocyte count Leukocyte differential Reticulocyte count Platelet count Morphologic assessment erythrocytes, leukocytes and platelets

Clinical chemistries Total Protein Albumin Urea Nitrogen (BUN) Creatinine Alanine Aminotransferase (ALT) Sorbitol dehydrogenase (SDH) Alkaline Phosphatase (ALP) Total Bile Acids Glucose Creatine Kinase (CK) Cholesterol Triglycerides

The results of all automated measurements for clinical pathology (unaudited data) shall be reported to the NTP within seven calendar days after sample collection.

18 ATTACHMENT 6

12.4.2 Blood For Micronuclei

Samples of blood (~200 l) shall be collected in EDTA from Core rats at termination. Samples are to be refrigerated immediately after collection and remain refrigerated until shipped the day of collection. The samples shall be shipped refrigerated (not frozen) for overnight delivery to an NTP-designated laboratory for micronuclei determination.

12.4.3 Mammary Glands

Clinical Pathology Group Males and Females: On PND 42 after blood collection, inguinal mammary glands shall be collected as per the NTP Specifications for Repro Studies. These animals shall be given a gross examination and tissues with gross lesions (and comparative controls) shall be fixed and retained for subsequent histopathological evaluation. For costing assume 2 animals/sex/group have a gross lesion (uterine or testicular).

12.4.4 SCVCE

SCVCE shall be conducted on Core subchronic (13 week) toxicity study rats in accordance with the protocol in the Appendix to the Specifications for Reproductive and Developmental Toxicity Studies. Vaginal cytology shall be performed starting on PND 77± 3 days, continuing for 16 days; sperm analyses shall be conducted.

12.4.5 Necropsy and Histopathologic Evaluation (PND 112+4)

Termination shall occur 13 weeks after weaning. Organ weights shall be determined from all animals surviving until the end of the study. Those organs to be weighed are: Liver, thymus, right kidney, left kidney, left testes, left epididymides, seminal vesicles w/coagulating glands, left ovary, right ovary, heart, prostate, and lungs. Organs shall be weighed to the nearest 10.0 mg except for testes, prostate, ovaries, and thymus that shall be weighed to the nearest 1.0 mg, and epididymides which shall be weighed to the nearest 0.1 mg.

A complete necropsy shall be performed on all treated and control animals that either die or are euthanized. All required tissues as listed below shall be saved, trimmed, embedded, sectioned and stained with hematoxylin and eosin for possible histopathologic evaluation. This shall be done for all animals in all groups.

Gross lesions shall be examined in all animals in all treatment and control groups. A complete histopathologic evaluation shall be done on all control animals, all animals in the highest treatment group with at least 60% survivors at the time of euthanasia, plus all animals in higher treatment groups.

Treatment-related lesions (target organs) shall be identified and examined in lower treatment groups to a no-effect level. For all natural death/moribund euthanasia animals a complete histopathologic evaluation shall be performed.

LIST OF TISSUES FOR COMPLETE HISTOPATHOLOGIC EVALUATION

Adrenal glands

Brain (7 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum

Ovaries

19 ATTACHMENT 6

and pons) Clitoral glands Pancreas

Esophagus Parathyroid glands

Eyes Pituitary gland

Femur (including diaphysis with marrow cavity and epiphysis (femoral condyle with epiphyseal cartilage plate, articular cartilage and articular surface)

Preputial glands

Gross lesions Prostate

Harderian glands Salivary glands

Heart and aorta Seminal vesicles (w/coagulating glands)

Intestine, large (cecum, colon, rectum) Spinal cord (3 sections if neurologic signs were present)

Intestine, small (duodenum, jejunum, ileum) Spleen

Kidneys Stomach (forestomach and glandular)

Testes with epididymides and vaginal tunics of testes

Liver (2 sections including left lateral lobe and median lobe) Thymus

Lungs and mainstem bronchi Thyroid gland

Lymph nodes-mandibular and mesenteric Tissue masses

Mammary gland with adjacent skin Trachea

Muscle, thigh (if neuromuscular signs were present)

Urinary bladder

Nasal cavity and nasal turbinates (3 sections) Uterus and vagina

Nerves, Sciatic Tibial and Trigeminal with ganglion (if neurologic signs were present)

12.4.6 Liver tissue for potential molecular analyses

Collect approximately 200 mg of tissue (as aseptically as possible), mince on an ice cold surface and flash freeze in cryotubes; store at a temperature of at least -70 ± 3oC. This tissue must be collected and frozen within 5 minutes of euthanasia. Samples shall be shipped to an NTP designated laboratory.

12.4.7 Urinalysis

Urine analyses shall be performed in F1 male and female rats PND 105 +4 (a 16-hour urine collection).

Appearance

Volume

Specific gravity

Microscopic assessment of sediment

Protein

Glucose

Creatinine

Activities of urine enzymes: N acetyl- B-glucosaminidase, lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase and gamma glutamyl transferase

12.5. REPORTING

20 ATTACHMENT 6

13. ONE-YEAR TOXICITY COHORT

13.1. ALLOCATION

F1 offspring shall be randomly allocated to the One-Year Toxicology cohort as described in Section 6.6 along with appropriate sentinel animals.

13.2. DIET

For animals in the One-Year cohort, the diet shall be NTP-2000 starting after weaning.

13.3. IN-LIFE ENDPOINTS

Out of cage clinical observations weekly; cage-side observations twice daily (before 10AM and after 2PM) including signs of, moribundity and/or death;

weekly body weights cage feed consumption (measured for a 7-day period) determined at 4 week intervals.

balano-preputial separation (BPS) PND 35 until acquisition (the COR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition.

Females: day of vaginal opening (VO) PND 23 until acquisition (the COR shall be consulted if VO has not occurred by PND 42) and body weight on day of acquisition; estrous cyclicity (starting at PND 77 ± 3 days; continuing for 16 days).

13.4. NECROPSY AND HISTOPATHOLOGIC EVALUATION (PND 386+4)

Animals shall be euthanized approximately one year after weaning. Organ weights shall be determined from all animals surviving until the end of the study. Organs to be weighed are: Liver, thymus, right kidney, left kidney, left testes, left epididymides, seminal vesicles w/coagulating glands, left ovary, right ovary, heart, prostate, and lungs. Organs shall be weighed to the nearest 10.0 mg except for testes, prostate, ovaries, and thymus that shall be weighed to the nearest 1.0 mg and epididymides that shall be weighed to the nearest 0.1 mg.

A complete necropsy shall be performed on all treated and control animals that either die or are euthanized. All required tissues shall be saved, trimmed, embedded, sectioned and stained with hematoxylin and eosin for possible histopathologic evaluation. This shall be done for all animals in all groups.

Gross lesions shall be examined in all animals in all treatment and control groups. A complete histopathologic evaluation shall be performed on all animals, including all natural death/moribund euthanasia animals.

LIST OF TISSUES FOR COMPLETE HISTOPATHOLOGIC EVALUATION

Adrenal glands

Brain with olfactory bulbs (7 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons)

Ovaries

Clitoral glands Pancreas

Esophagus Parathyroid glands

Eyes Pituitary gland

21 ATTACHMENT 6

Femur (including diaphysis with marrow cavity and epiphysis (femoral condyle with epiphyseal cartilage plate, articular cartilage and articular surface)

Preputial glands

Gross lesions Prostate

Harderian glands Salivary glands

Heart and aorta Seminal vesicles (w/coagulating glands)

Intestine, large (cecum, colon, rectum) Spinal cord (3 sections if neurologic signs were present)

Intestine, small (duodenum, jejunum, ileum) Spleen

Kidneys Stomach (forestomach and glandular)

Testes with epididymides and vaginal tunics of testes

Liver (2 sections including left lateral lobe and median lobe) Thymus

Lungs and mainstem bronchi Thyroid gland

Lymph nodes-mandibular and mesenteric Tissue masses

Mammary gland with adjacent skin Trachea

Muscle, thigh (if neuromuscular signs were present)

Urinary bladder

Nasal cavity and nasal turbinates (3 sections) Uterus and Vagina

Nerves, Sciatic, Tibial and Trigeminal with ganglion (if neurologic signs were present)

13.5. LIVER SECTIONS FOR POTENTIAL MOLECULAR ANALYSES

Collect approximately 200mg of tissue (as aseptically as possible), mince on an ice cold surface and flash freeze in cryotubes; store at a temperature of less than -70 +/- 3oC. This tissue must be collected and frozen within 5 minutes of euthanasia. Tissues shall be shipped to an NTP designated laboratory.

13.6. REPORTING

14. MATERNAL TRANSFER STUDY

14.1. ALLOCATION AND OVERVIEW

A separate group of animals shall be used to evaluate gestational and lactational transfer of the test article to the offspring at GD 18 and PND 4. After the chemistry methods are developed from tissues and plasma collected in the dose range-finding study, the COR shall notify the Contractor to proceed with the maternal transfer study.

Time mated rats (same strain, supplier and diet as used in the MOG) shall be obtained from the supplier and administered the test article at doses of 100, 500 and 1000 mg/kg/day by gavage daily starting on GD 6. There shall be 12 pregnant dams/dose.

14.2. IN-LIFE ENDPOINTS

(before 10AM and after 2PM) including signs of delivery, moribundity and/or death;

Body weights recorded daily and feed consumption measured every three days.

Observations for pups and maternal behavior.

22 ATTACHMENT 6

14.3. PROCEDURES

On GD 18, 6 dams from each dose group shall be euthanized by decapitation and the following collected:

Maternal blood –obtain plasma (at least 3 ml plasma…