ATTACHMENT 3_SOW-APPENDIX 2.pdf

APPENDIX 2 – MOG AND RACB TEMPLATES

MOG_current_508.doc

ATTACHMENT 3

Instructions: input or delete (as appropriate) information in bold underlined sections

PROTOCOL OUTLINE FOR THE MODIFIED ONE GENERATION STUDY (MOG,

RXXXX) OF TEST ARTICLE (TA abrv; CAS XXX-X-X) IN HARLAN SPRAGUE DAWLEY RATS ADMINISTERED VIA DOSED FEED or DAILY IN/BY ROUTE OF EXPOSURE (if gavage/other)

Study Scientist: insert name, add project leader if not the same person

Nomination: insert general nomination information

Contract Lab: insert lab

1. INTRODUCTION:

1.1. Background and Rationale insert detailed nomination information and assessment strategy

2. OBJECTIVE: To characterize the reproductive toxicity of TEST ARTICLE in Harlan

Sprague Dawley rats using the modified one-generation (MOG) study design Add others aspects as appropriate-(nonstandard items; TK, ADME etc.)

The MOG is composed of two interrelated parts: (1) if the acceptable range of dose levels required to avoid excessive general and perinatal toxicity is unknown, a dose range-finding (DRF) study is conducted to determine the maximal dose level that is tolerated by the dam (dosing gestation day 6 through weaning) and that has no impact on perinatal survival until weaning (2) in the definitive MOG, sperm (+) females are administered the test material from gestation day 6 through weaning. The subsequent F1 offspring are then continuously administered the test article via the same route of exposure as the dams. All F1 animals after PND 4 are taken to adulthood for gross and histo-pathology examination and can be allocated to various cohorts or “cassettes” (e.g. prechronic, mating to assess fecundity/fertility, teratology, neuro- or immuno-endpoints).

NOTE- Sections 3 and 4 apply to both the DRF and the MOG definitive study

3. TEST MATERIAL

3.1. Structure (if applicable)- describe source, components [major and active/marker for chemical analyses])

3.2. Molecular Formula:

3.3. Molecular Weight:

3.4. CAS #:

3.5. Characterization: Prior to study start, and every 24 +/- 2 weeks until euthanization of the last animal in the MOG definitive study, a one gram sample each of the bulk material and frozen reference shall be submitted for analysis by an NTP designated laboratory for the purpose of confirming purity of the bulk material relative to the frozen reference. In addition, 50 mL representative samples shall be submitted for concentration determination to the same NTP-designated laboratory from the first set of formulations prepared for the study and from the animal room on the last dosing day using this formulation set.

Formulation and animal room samples shall be submitted for analysis again from the projected middle and last formulation set of the study. - (include until NTP repro specs are complete; also include homogeneity if suspension)

3.6. Route of Administration oral Feed or Gavage or Drinking water (include dose volume e.g. 2 or 5 mL/kg) or (other)

3.7. Vehicle information (Milled feed; irradiated and certified diet or w/ CAS #, if appropriate)

4. TEST ANIMALS

4.1. Species, Strain/Stock and Age: Time-mated (assumed pregnant) female Harlan

Sprague Dawley Rats (Harlan Laboratories) (or other). F0 female rats shall be sexually mature (11-12 weeks; 200-225 g) and mated at the supplier. Male (and female) F1 rats shall be sexually mature when mated.

4.2. Diet and Water: Tap water and Milled/pelleted Feed (NIH-07; from an NTP-approved vendor, or other diet e.g. low phytoestrogens diet; include rationale for different diet) to be provided ad libitum.

4.3. Pre-study evaluation: Animals shall be obtained on gestation day (GD) 2. Day of positive evidence of mating = GD 0. Animals will be examined by the veterinarian before being released for study. A COTR-approved screening program shall be followed to ensure the health of the animals on study. A sufficient number of animals shall be purchased for this purpose.

4.4. Randomization and Identification: Prior to dosing/administration of dosed feed (e.g. GD 3), F0 female rats shall be randomized into treatment groups 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. 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 COTR.

5. PROCEDURES

5.1. Study Design: Dose Range-Finding Study (DRF) (if required to set dose levels for the definitive MOG study); also consider adding additional rats for biological sampling.

5.1.1. Caging

Dams shall be housed one per cage (except for dams with pups) by dose group. Pups shall remain with their respective dam until euthanasia on

PND 21.

5.1.2. Dose levels and number of rats

Rats shall be randomized as described in Section 4.4 into 6 groups and continuously exposed via feed (or gavage) containing the ppm indicated below:

Number of F0 females may be increased to accommodate TK/biological sampling.

5.1.3. Duration of dosing (and frequency if gavage)

Females shall be administered dosed feed/ dosed daily from GD 6 through postnatal day (PND) 21 (i.e. up to ~36 days or longer). PND 0 is defined as the day that all pups within a litter have been delivered. Note- the Contract Laboratory may define PND 0 differently; subsequent intervals may need to be adjusted to accommodate For gavage studies, dose volume will be based on the most dam and pup recent body weight.

Note- pup dosing would begin on PND 12.

5.1.4. Antemortem endpoints

Out of cage observations once daily (for gavage studies this should occur 1-3 hrs post dose); cage side clinical observations, twice daily (before 10AM and after 2PM) including signs of delivery, moribundity

Group Dose levels (mg/kg, ppm, etc)

No. of F0 females

Control (1) 8

2 8

3 8

4 8

5 8

6 8

Total 48 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 daily recorded during gestation (day of randomization onwards); PND 1, 4, 7, 14 and 21(for gavage studies daily). 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, 7-14, 14-21, PND 1-21.

Dam feed consumption measured for (include water consumption- if TA is administered in water) GD 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6- 21, and PND 1-4, 4-7, 7-14, 14-21, 1-21.

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.

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

5.1.5. Biological sample collection for method validation (if conducted)

On GD 18, confirmed pregnant dams (3 dams/dose group at 0, XXXXand XXXXXppm) shall be euthanized and the following collected:

Maternal plasma isolated from trunk blood (at least 3.0 ml [target]; stored at -70oC).

Amniotic Fluid (pooled by litter; at least 1ml [target]; stored at - 70oC) Fetuses (pooled by litter, frozen in liquid nitrogen and stored at - 70oC)

On PND 4, dams and their pups (3 dams/group at 0, 2250 and 20,000 ppm) shall be euthanized and the following collected:

Maternal Plasma (at least 3.0 ml [target]; stored at -70oC) Whole pup in a manner appropriate for chemical analysis (3 per sex/litter)

Tissues and plasma shall be sent to the designated NTP Chemistry contractor for analyses.

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

5.1.6. Euthanasia

Rats shall be euthanized by Contractor-IACUC-approved procedures acceptable to the NTP COTR. 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 will be discarded without examination. Rats that do not appear to be pregnant and do not deliver will be examined for the presence of implants (using ammonium sulfide, or Prussian blue, or other appropriate technique) and corpora lutea enumerated.

5.2. Study Design: Definitive Study

5.2.1. Caging

Dams shall be housed one per cage (except when with their pups) by dose group. Pups shall remain with their respective dam until weaning on PND 21 (or euthanasia). After weaning, juveniles shall be group housed by litter and sex (up to 4 per cage as appropriate for their size). When in co-habitation, rats shall be housed as pairs of one male and one female per cage from the same dose group (up to 15 days, or until positive evidence of mating). After evidence of mating, or after 15 days if no evidence of mating, the pairs will be separated and individually housed until necropsy (delete if F1 mating will not be conducted).

5.2.2. Dose levels and number of animals

Rats shall be randomized as described in Section 4.4 into 4 groups:

Group Dose levelsa (mg/kg, ppm, etc)

No. of F0 females

Vehicle Control

0 25

Low Dose TBD 25

Mid-Dose TBD 25

High-Dose TBD 25

Total F0 100 a: F1 offspring shall also be administered the test article post-weaning at the same dose as their parents

5.2.3. Duration of dosing

F0 rats shall be administered control- or dosed-feed ad libitum (or water) containing the test article or dosed daily by gavage- include dose volume [e.g. 2 or 5 mL/kg]), 7 days a week, from GD 6 throughout gestation, lactation and continuing until necropsy.

F1 generation pups shall be exposed via nursing/eating dosed feed. After weaning the offspring shall be administered control- or dosed feed (at the same dose level the dam received) ad libitum, 7 days a week until euthanasia. Rats allocated to a prenatal toxicity or fertility cohort will be administered the test compound in the diet during breeding and gestation until scheduled necropsy. For gavage studies, dose volume will be based on the most dam and pup recent body weight. Note- pup dosing would begin on PND 12.

Phase of study Total days on feed

F0 females-until weaning

~36 days

F1 Toxicology cohort

~91 days

F1 Teratology cohort up to 106 days

F1 Breeding cohort up to 136;

up to 164 w/crossover

F1 Neurotox cohort

TBD ~91 days

F1 Immunology cohort

~35 days + time at immunotox contractor

5.2.4. F1 production, randomization and disposition

F1 pups shall be uniquely identified on PND 1 by paw tattoo. When possible, F1 litters shall be standardized to 8 pups (4 male and 4 females) on PND 4 (or 10 pups (5+5) if all cohorts are conducted). Pups will be randomly allocated (by treatment) to the following cohorts on PND X (depends on cohorts conducted); provide appropriate text to indicate which cohort take priority:

Potential 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

Toxicity 1 male and 1 female

10 10 80

Prenatal Toxicology

1 male and 1 female

20 20 160

Fertility 1 male and 1 female

20 20 160

Developmental Neurotoxicology

1 male and 1 female

20 20 160

Immuno toxicity 1 male and 1 female

20 20 160

* Extra offspring from each of these cohorts may be utilized for or be used for “special studies” (i.e. clinical pathology, TK/ADME, etc).

Note- if additional litters are available or if all cohorts are not conducted, then the number of pups allocated to other groups may be increased.

Inguinal mammary glands from up to 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. See Section 5.2.7 for post weaning mammary gland collection.

Non-selected F1 pups shall be euthanized on PND 4. All pups and rats at necropsy shall be euthanized by a Contractor-IACUC-approved procedures acceptable to the NTP COTR. Note- if it is not possible to determine sex on PND 4 (i.e. apparent skewed sex ratio), culling may need to be delayed until weaning.

5.2.5. Antemortem endpoints (applicable to all cohorts, as appropriate)

5.2.5.1. F0 dams

Out of cage observations once daily; cage-side clinical 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 (day of randomization onwards) and PND 1, 4, 7, 10, 13, 16, 19 and 21 (for gavage studies daily); 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; PND 1-16; 1-

21, 4-21

Feed consumption (and water consumption-if administered in the water); 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 and PND 1-16; 1-21, 4-21

Number of pregnant females and duration of pregnancy.

5.2.5.2. F1 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).

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 and 21 (and PND 28 if weaning is delayed) (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 4-21

Males: Presence of areolae on PND 13, testicular descent (PND 14-30), balano-preputial separation ((BPS); PND 35 until acquisition; the COTR 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 COTR shall be consulted if VO has not occurred by PND 42) and body weight on day of acquisition and estrous cyclicity (starting at PND 77± 3 days and continuing for 16 days).

After weaning: out of cage observations once daily; cage-side clinical observations, twice daily (before 10AM and after 2PM) including signs of, moribundity and/or death; weekly body weights and cage feed consumption (and water consumption if the TA is supplied in the water)

5.2.6. F0 Necropsy

F0 rats shall be necropsied after weaning has been completed.

Animals will be subjected to external examination and uterine implantation scars enumerated. Gross lesions and representative control tissues will be retained, processed to slides and examined histopathologically. Rats that do not appear to be pregnant and do not deliver will be examined for the presence or evidence of implants (using ammonium sulfide or Prussian blue, or other appropriate technique) and corpora lutea enumerated.

THE FOLLOWING SECTIONS INVOLVE MULTIPLE COHORTS

1.1.1. (POST) WEANING MAMMARY GLAND; BIOLOGICAL SAMPLING

COHORT

1.1.1.1. Allocation

F1 offspring shall be randomly allocated to the post weaning mammary gland; biological sampling cohort as described in Section 5.2.4.

1.1.1.2. Antemortem Endpoints

Endpoints identified Section 5.2.5.2, except assessments for BPS, vaginal opening (other than rats that shall be assessed for mammary gland development) and vaginal cytology.

1.1.1.3. Post weaning Mammary Gland Analyses

The inguinal mammary glands from one F1 offspring/sex/litter at the time of vaginal opening (female) or PND 21 (male) shall be collected and whole mounts prepared (up to ten/sex/dose level at the appropriate interval). 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.

1.1.1.4. Biological Sampling

F1 male and female rats allocated to the biological sampling cohort shall continue to receive their respective test diet. On PND 21 and 56, 5 animals/sex from each dose group shall be euthanized and the following collected:

Plasma isolated from trunk blood (at least 1.5 ml [target]; stored at -70oC), kidneys (paired), epididymides (paired), testes (paired), ovaries (paired), and liver (left lateral lobe; ~1g); flash frozen and stored at -70oC.

Plasma and tissues shall be sent to the designated NTP Chemistry contractor.

1.1.1. TOXICITY COHORT

F1 offspring will be randomly allocated to the Toxicology cohort as described in Section X. If additional Clinical Pathology endpoints (or other) are collected, additional “special study rats” will need to be allocated (from the unallocated toxicity litters). These will be different from the “core” toxicity rats.

NOTE: Clinical observations are not required for special study rats; however, body weights are to be measured for calculation of dose.

1.1.1.2. Antemortem Endpoints

See Section 5.2.5.2

1.1.1.3. Toxicological Parameters to be Evaluated: [Include the following as appropriate for the test article under study]

F1 Clinical Laboratory Studies Rats shall be bled for the terminal collection for clinical laboratory studies.

Additional special study rats (other than the 10 core study rats) shall be dosed, both sexes, all treatment and controls groups, for collections at days PND 24 ± 2 and PND 42 ± 4 (or other).

Blood shall be collected from the retro-orbital sinus under CO2/O2 anesthesia at days 24 ± 2 & 42 ± 4 from the special study rats and these animals shall be sacrificed after the collection on PND 42 with no further evaluation. The core study rats shall be bled for the terminal collection for clinical laboratory studies. Core study rats shall be bled at terminal euthanasia for hematology determinations.

All male and female animals shall be treated for approximately the same number of days (PND 112 ± 4) before collection of samples, and all animals of a sex shall be bled on the same day.

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, for the early time points (if conducted) and within 21 days for measurements conducted at terminal sacrifice.

Blood For Micronuclei

Samples of blood (~200 l) shall be collected in EDTA from 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.

Mammary Glands The inguinal mammary glands from one F1 offspring/sex/litter at the time of vaginal opening (female) or PND 21 (male) shall be collected and whole mounted (up to ten/sex/dose level at the appropriate interval). 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. Note- this can only be conducted if extra animals are available.

SCVCE

SCVCE will be conducted on [core] toxicity study rats in accordance with the protocol in the Appendix to the Specifications. The NTP Project Officer shall be notified by PND 70 of the study regarding mortality, body weight changes and clinical signs of toxicity for use in determining the treatment groups to be used in the SCVCE. Vaginal cytology will be assessed on all cohorts (as applicable); sperm analyses will not need to be conducted if it is assessed in the fertility and/or prenatal cohorts

Necropsy and Histopathologic Evaluation (PND 112+4)

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, testes, epididymides, seminal vesicles w/coagulating glands, ovaries, heart, prostate, and lungs. Organs shall be weighed to the nearest 10.0 mg except for testes, epididymides, prostate, ovaries, and thymus that shall be weighed to the nearest 1.0 mg (See NTP 2011 Specifications).

A complete necropsy shall be performed on all treated and control animals that either die or are euthanized, and all tissues as listed in NTP 2011 Specifications shall be saved. All tissues required for complete histopathology as listed in the NTP 2011 Specifications shall then be 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 Mammary gland with adjacent skin Brain (7 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons)

Muscle, thigh (if neuromuscular signs were present)

Clitoral glands

Nasal cavity and nasal turbinates (3 sections)

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

Parathyroid glands

Gross lesions Pituitary gland Harderian glands Preputial glands Heart and aorta Prostate Intestine, large (cecum, colon, rectum) Salivary glands Intestine, small (duodenum, jejunum, ileum) Seminal vesicles (w/coagulating glands) Kidneys Gallbladder (mouse)

Spinal cord, sciatic and trigeminal nerves

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

Spleen

Lungs and mainstem bronchi Stomach (forestomach and glandular) Lymph nodes - mandibular and mesenteric Testes with epididymides Thymus Thyroid gland Tissue masses

Trachea

Urinary bladder Uterus

Liver sections for potential molecular analyses (provide detail as to location and amount)

Urinalysis (OPTIONAL NOT PART OF STANDARD DESIGN) 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

[Include only those assays below that are appropriate for the test article under study] N-acetyl-B-glucosaminidase (NAG) Lactate dehydrogenase (LDH) Alkaline phosphatase (AP) Aspartate aminotransferase (AST)

1.1.1. PRENATAL TOXICOLOGY COHORT; Note- if not conducted the number of animals allocated to the fertility cohort would be doubled)

F1 offspring shall be randomly allocated to the prenatal toxicity cohort as described in Section 5.2.4. Vaginal cytology will be initiated on week 11 (prior to cohabitation). After 16 days (week 13), one male and one female from the same dose group, and avoiding sibling mating, will be cohabitated. After evidence of mating (or after 15 days if no evidence of mating), male and females shall be separated and females will be terminated on

GD 21.

1.1.1.2. F1 Antemortem endpoints (males and females)

Endpoints identified in Section 5.2.5.2)

F1 Dams

Time to mating (pre-coital interval)

Out of cage observations once daily; cage-side clinical 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

1.1.1.3. F1 Dam Necropsy

The following endpoints shall be measured:

Maternal body, liver, adrenal, ovarian, 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 examined for the presence or evidence of implants (using Prussian Blue or other appropriate method).

All live fetuses shall be counted, weighed, sexed (visual anogential distance). All fetuses (and late resorptions to the extent possible) shall be examined for external morphological abnormalities, including cleft palate. For each dam, the position of each fetus in the uterine horns, starting at the left end, noting the cervix and moving to the right end, shall be noted. The placentas from live fetuses shall undergo gross observation.

Fetuses shall be euthanized and 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.

1.1.1.4. F1 Parental Necropsy

Males

Males will 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 Ovaries, right and left

Testis, right and left Preputial glands a

Seminal Vesicles (w/ Coagulating Glands)

Levator ani bulbocavernosus a: Only be collected and weighed when an antiandrogenic effect is expected (e.g. observed increased incidence of areolae, delay in PPS)

1.1.1. FERTILITY COHORT

F1 offspring shall be randomly allocated to the Fertility cohort as described in Section X. Vaginal cytology will be initiated on PND 77+ 3 (prior to cohabitation). After 16 days, one male and one female from the same dose group, and avoiding sibling mating, will be cohabitated (up to 40 pairs/dose; i.e. two males and two females from each litter). After evidence of mating (or after 15 days if no evidence of mating), male and females shall be separated and females shall be allowed to litter. Pups will remain with their dams until weaning.

1.1.1.2. Antemortem endpoints

Endpoints identified in Section 5.2.5.2

F1 dams

Time to mating (pre-coital interval)

Out of cage observations once daily; cage-side clinical 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 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 (and water consumption-if administered in the water) 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 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 and 21 (and PND 28 if weaning is delayed) (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 4-21

Males: Presence of areolae on PND 13. If the male pups are retained past PND 21 (e.g. evidence of reproductive toxicity) and at the direction of the COTR, than testicular descent (PND 14-30), balano-preputial separation (BPS; PND 35 until acquisition; the COTR shall be consulted if BPS has not occurred by PND 50) and body weight on day of acquisition shall be collected.

Females: If the female pups are retained past PND 21 and at the direction of the COTR, day of vaginal opening (PND 23 until acquisition; the COTR shall be consulted if VO has not occurred by PND 42) and body weight on day of acquisition and estrous cyclicity (PND 77 to PND 93).

1.1.1.3. F1 Parental Necropsy

Females shall be necropsied after the F2 offspring have been euthanized

Males shall be necropsied after potential effects on fertility have been ascertained (see note 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.

Trunk blood shall be collected and sera isolated (target volume 2mL) and frozen for potential hormone (or other potential) analyses as directed by the COTR

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.

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 gland

Levator ani bulbocavernosus a: Only be collected and weighed when an antiandrogenic effect is expected (e.g. decrease in AGD, observed increased incidence of areolae, or a delay in PPS)

Fix and process the following tissuesa per established NTP specifications:

Adrenal Glands Ventral Prostate

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

Ovaries c

Epididymis, right b Pituitary

Testis, right b Thyroid Gland

Seminal Vesicles (w/ Coagulating Glands)

Gross lesions

Dorsal Prostate Nipple/adjacent mammary gland d 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 See Section 5.2.9.3 c: Step sectioned and primordial follicles enumerated d: One retained nipple from each male with retained nipples shall be collected for histopathologic evaluation.

Fixed tissues will be processed to slides and stained with hemotoxylin and eosin. The high 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 COTR.

1.1.1.4. F2 Necropsy

Pups shall be necropsied on PND 21 and subjected to an external and internal examination. All gross lesions and comparative controls will be retained and processed for histopathological examination.

NOTE- Determination of Affected Sex:

Determination of Affected Sex – Crossover Mating

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 COTR, the Contractor shall conduct a cross-over mating. 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 mated with the high dose males when they are sexually mature at 11 weeks of age.

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 COTR that no further testing of them (such as dominant lethal test) is required.

1.1.1. NEUROBEHAVIORAL COHORT (will “follow” OPPTS 870.6300)

F1 offspring will be randomly allocated to the Toxicology cohort as described in Section X.

1.1.1.2. Antemortem endpoints

See Section 5.2.5

1.1.1.3. Motor activity

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

1.1.1.4. Auditory startle test

An auditory startle habituation test should be performed on the offspring around the time of weaning and around day PND 60.

1.1.1.5. Learning and memory test

A test of associative learning and memory should be conducted around the time of weaning and around PND 60.

1.1.1.6. Neuropathology.

Neuropathological evaluation should be conducted on animals on postnatal day 11 and at the termination of the study. At 11 days of age, one male or female pup should be removed from each litter such that equal numbers of male and female offspring are removed from all litters combined. Of these, six male and six female pups per dose group will be sacrificed for neuropathological analysis. The pups will be killed by exposure to carbon dioxide and immediately thereafter the brains should be removed, weighed, and immersion-fixed in an appropriate aldehyde fixative. The remaining animals will be sacrificed in a similar manner and immediately thereafter their brains removed and weighed. At the termination of the study, one male or one female from each litter will be killed by exposure to carbon dioxide and immediately thereafter the brain should be removed and weighed.

In addition, six animals per sex per dose group (one male or female per litter) should be sacrificed at the termination of the study for neuropathological evaluation.

1.1.1. IMMUNOTOX COHORT

Animals shall be allocated as outlined in Section 5.2.4 and transferred on PND 25 to an NTP designated contractor with sufficient amount of control and dosed feed to last until PND 50. Study scheduling and animal shipment must be coordinated with the 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. The Immunotoxicology Discipline Leader or a designee will provide a separate stand-alone study protocol for the Evaluation of Immunotoxicity to the Project Approval Committee.

1.1. MATERNAL TRANSFER STUDY

A separate study shall evaluate gestational and lactational transfer of EHMC to the offspring at the GD 18 and PND 4 time points. After the chemistry methods are developed from tissues collected in the dose range finding study, the COTR shall notify the Contractor to proceed with the maternal transfer study. Time mated rats (same strain and diet as used in the MOG) shall be administered EHMC in the feed at doses TBD (10 dams per dose, 3 dose levels), starting on GD 6.

On GD 18, 5 dams from each dose group shall be euthanized by decapitation and the following collected:

Maternal plasma (at least 3 ml [target]), Fetuses (pooled by litter), Amniotic Fluid (pooled by litter, minimum of 1 ml [target])

The remaining 5 dams per dose group shall continue to be dosed until PND 4. On PND 4, these dams shall be euthanized by decapitation and the following collected:

Maternal Plasma (at least 3.0 ml [target]), Whole pup (3 per sex)

6. Data Presentation (WILL HAVE TO BE UPDATED TO REFLECT CHANGES IN

ENDPOINTS)

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

• Mean body weights, body weight gains, food and water consumption (see Sections 5.1.5 and 5.2.5)

• Male and female fecundity and fertility indices

• 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

• AGD (by sex, nested by litter and co-varied by body weight)

• Mean number of areolae and nipples (by litter)

• Organ weights

Note- Summaries of data shall be provided to the COTR within five days after each of the following:

End of the DRF study (including dose recommendations for the RACB study);

Delivery of the last individual dam for each F1 and F2 litter (i.e. one report for the F1a litter, another report for the F1b litter, etc.); and

Acquisition of BPS and VO by all pups in the F1c (or PND 50 if either has not occurred).

7. TRACKING ACTIVITY OF PROJECT

The computer system used to keep record of all data related to the project shall be approved by the NTP-COTR.

8. PROTOCOL CHANGES

Prior to changes in the protocol, the NIEHS COTR shall be consulted and agreed to changes in writing. The change and the reason for the change shall be put in writing in the form of an amendment, signed by both the contract lab Study Director and the NTP COTR, and maintained with the original approved protocol.

Additional Points to consider during protocol generation (will be templated in the future) Addition of neuro-behavioral

RACB_508.DOC

Instructions: input or delete (as appropriate) information in bold underlined sections

PROTOCOL OUTLINE FOR THE NTP REPRODUCTIVE ASSESSMENT BY

CONTINUOUS BREEDING (RACB, RXXXX) OF TEST ARTICLE (TA abrv; CAS XXX-X- X, include NTP Number) IN HARLAN SPRAGUE DAWLEY RATS ADMINISTERED VIA

DOSED FEED or DAILY IN/BY ROUTE OF EXPOSURE (if gavage/other)

Study Scientist: insert name, add project leader if not the same person

Nomination: insert general nomination information

Contract Lab: insert lab

1. INTRODUCTION:

1.1. Background and Rationale insert detailed nomination information and assessment strategy

2. OBJECTIVE:

To characterize the reproductive toxicity of TEST ARTICLE in Harlan Sprague Dawley rats using the NTP RACB design Add others aspects as appropriate-(nonstandard items; TK, ADME etc.)

The RACB is composed of four interrelated parts: (1) if the acceptable range of dose levels required to avoid general toxicity is unknown, a dose range-finding (DRF) study is conducted; (2) cohabitation of F0 males and females to produce three (3) F1 generation litters for assessment, with chemical administered continuously from pre-cohabitation of F0 animals and weaning of F1 animals until their necropsy; (3) if an effect on the fertility or fecundity of F0 animal is seen, a crossover mating of F0 high-dose, exposed males with näive, unexposed females and high-dose, exposed females with control males shall be conducted to determine the affected sex(es); and (4) cohabitation of F1 males and females to produce three (3) F2 generation litters for assessment, with chemical administered continuously to F1 animals during cohabitation and to F2 animals from weaning until their necropsy.

NOTE- Sections 3 and 4 apply to both the DRF and the RACB main study

3. TEST MATERIAL

3.1. Structure: (if applicable)- describe source, components [major and active/marker for chemical analyses])

3.2. Molecular Formula:

3.3. Molecular Weight:

3.4. CAS #:

3.5. Characterization: Prior to study start, and every 24 +/- 2 weeks until euthanization of the last animal in the RACB definitive study, a one gram sample each of the bulk material and frozen reference shall be submitted for analysis by an NTP designated laboratory for the purpose of confirming purity of the bulk material relative to the frozen reference. In addition, 50 g/mL representative samples shall be submitted for concentration determination to the same NTP-designated laboratory from the first set of formulations prepared for the study and from the animal room on the last dosing day using this formulation set.

Formulation and animal room samples shall be submitted for analysis again from the projected middle and last formulation set of the study. - (include until NTP repro specs are complete; also include homogeneity if suspension)

3.6. Route of Administration oral Feed or Gavage or Drinking water (include dose volume e.g. 2 or 5 mL/kg) or (other)

3.7. Vehicle information (Milled feed; irradiated and certified diet or w/ CAS #, if appropriate)

4. TEST ANIMALS

4.1. Species, Strain/Stock and Age: Male and Female Harlan Sprague Dawley Rats (Harlan Laboratories) (or other). F0 female and male rats shall be sexually mature at the start of dosing (at 11-12 and 13-14 weeks of age, respectively).

Rats of each sex should be within +5% of the respective mean weight when shipped from the vendor.

4.2. Diet and Water: Tap water and Milled/pelleted Feed (NIH-07; from an NTP-approved vendor, or other diet e.g. low phytoestrogens diet; include rationale for different diet) to be provided ad libitum.

4.3. Pre-study evaluation: Animals shall be obtained 10-14 days prior to study start and undergo “standard quarantine” prior to use. An NTP- COTR-approved sentinel program shall be followed to ensure the health of the animals on study. A sufficient number of animals shall be purchased for this purpose.

4.4. Randomization and Identification: Prior to dosing/administration of dosed feed F0 rats shall be randomized into treatment groups by sex to minimize the variance in mean body weight, and uniquely identified by tail tattoo. F1c and F2c pups shall be identified by paw tattoo according to number within litter at PND 1 and, when retained beyond PND 21, uniquely identified at weaning.

4.5. 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 NTP COTR.

5. PROCEDURES

5.1. Study Design: Dose Range-Finding Study (DRF) (if required to set dose levels for the definitive MOG study); also consider adding additional rats for biological sampling.

5.1.1. Caging

Rats shall be housed up to five per cage (depending on cage size) by sex and dose group until they are paired for mating. During the mating cohabitation period, rats shall be housed one male and one female per cage from the same dose group (up to 15 days, or until positive evidence of mating). After evidence of mating, or after 15 days if no evidence of mating, the pairs shall be separated and individually housed (except for dams with pups) until necropsy. Pups shall remain with the respective dam until necropsy on PND 4.

5.1.2. Dose levels and number of rats

Rats shall be randomized as described in Section 4.4 into 6 groups and continuously exposed via feed containing the ppm indicated below: Number of F0 females may be increased to accommodate TK/biological sampling.

5.1.3. Duration of dosing (and frequency if gavage)

Males and females shall administered dosed feed/ dosed daily for 7 days (or 14 days- or longer depending on the chemical) a week from the pre-cohabitation dosing period, during the cohabitation breeding period, gestation, and until the rats and pups are euthanized on PND 4, as appropriate (i.e. up to 56 days or longer). PND 0 is defined as the day that

Group Dose levels (mg/kg, ppm, etc)

No. of F0 males

No. of F0 females

Control (1) 8 8

2 8 8

3 8 8

4 8 8

5 8 8

6 8 8

Total F0 48 48 all pups within a litter have been delivered. Note- the Contract Laboratory may define PND 0 differently; subsequent intervals may need to be adjusted to accommodate For gavage studies, dose volume shall be based on the most recent body weight.

5.1.4. Cohabitation

Male and female rats shall be cohabitated up to 15 days, or until positive evidence of mating. If after 15 days there is no evidence of mating, then the rats shall be separated and the females monitored for delivery up 23 days.

Day of positive evidence of mating = GD 0.

5.1.5. Antemortem endpoints

Time to mating (pre-coital interval)

Out of cage observations once daily (for gavage studies this should occur 1-3 hrs post dose); cage side clinical 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.

Male body weights twice weekly (NOTE- twice weekly is defined as every three, then 4 days).

Female body weights, twice weekly at all times during the study except during gestation when they shall be recorded daily; PND 1 and 4. Twice weekly weights are to be reported as well as the post-natal weights for the following intervals: pre-mating period, GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, and 0-21; PND 1-4.

Male feed consumption twice weekly (unless breeding). Female feed consumption twice weekly prior to mating, and for (include water consumption- if TA is administered in water) GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 0-21, and PND 1-4. Food consumption data shall not be collected during cohabitation.

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 and 4

Number of non-pregnant rats, duration of pregnancy

5.1.6. Biological sample collection for method validation (if conducted)

On GD 18, confirmed pregnant dams (3 dams/dose group at 0, XXXX and XXXXX ppm) shall be euthanized and the following collected:

Maternal plasma isolated from trunk blood (at least 3.0 ml [target]; stored at -70oC).

Amniotic Fluid (pooled by litter; at least 1ml [target]; stored at -

Fetuses (pooled by litter, frozen in liquid nitrogen and stored at -

On PND 4, dams and their pups (3 dams/group at 0, 2250 and 20,000 ppm) shall be euthanized and the following collected:

Maternal Plasma (at least 3.0 ml [target]; stored at -70oC) Whole pup in a manner appropriate for chemical analysis (3 per sex/litter)

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

6. Euthanasia

Rats shall be euthanized by a Contractor-IACUC-approved procedure acceptable to the NTP COTR. Males shall be euthanized after the cohabitation period and discarded without examination. Females and pups shall be euthanized on PND 4 (or sufficiently after expected littering date, if no evidence of pregnancy). Rats that produced litters shall be discarded without examination. Rats that do not appear to be pregnant and do not deliver shall be examined for the presence of implants (using ammonium sulfide or Prussian blue, or other appropriate technique) and corpora lutea enumerated.

6.1. Study Design: RACB

6.1.1. Caging

Rats shall be housed up to four per cage (depending on cage size) sex per cage until they are paired for mating. During the mating cohabitation periods, rats shall be housed as pairs of one male and one female per cage from the same dose group (up to 15 days, or until positive evidence of mating). After evidence of mating, or after 15 days if no evidence of mating, the pairs shall be separated and individually housed until cohabited again for production of the next litter or until necropsy. The same male and female shall be re-paired each time as “life-long mating pairs”. Pups shall remain with their respective dam until termination or weaning. After weaning, juveniles shall be group housed by litter and sex (2-3 per cage as appropriate for their size).

6.1.2. Dose levels and number of animals

Rats shall be randomized as described in Section 4.4 into 4 groups:

Group Dose levels

(mg/kg, ppm, etc) No. of F0 males No. of F0 females

Vehicle Control 0 22 22

Low Dose 22 22

Mid-Dose 22 22

High-Dose 22 22

Total F0 88 88 a: F1c and F2c offspring shall also be directly administered the test article post-weaning at the same dose as their parents

6.1.3. Duration of dosing

F0 rats shall be administered control- or compound-containing feed ad libitum continuously (or water) containing the test article or dosed daily by gavage- include dose volume [e.g. 2 or 5 mL/kg]), 7 days a week, from the pre-cohabitation dosing period (2 weeks prior to cohabitation for both males and females), throughout breeding and gestation, and until necropsy. F1 and F2 offspring shall be exposed via nursing.