DuPont Chemicals & Fluoroproducts 

Chestnut Run Plaza 702 1276-E 

4417 Lancaster Pike 

Wilmington, DE 19805 

USA 

aupoNTm 

DuPont Chemicals & Fluoroproducts 

September 17, 2012 

U.S. Environmental Protection Agency 

Office of Air and Radiation 

Stratospheric Protection Division 

Attention: Margaret Sheppard 

1310 L Street NW, iolh Floor (Room 1029) 

Washington, DC 20005 

Dear Ms Sheppard: 

Reference: DuPont SNAP Information Notice 

(Z)-1,1,1,4,4,4-hexafluoro-but-2-ene (CAS RN 69 2-49-9) 

Sincerely, 

Chestnut Run Plaza 702/1276-E, Wilmington, DE 19805 

E. I. du Pont de Nemours and Company 
SANITIZED SUBMISSION 

ATTACHMENT HEADER SHEET 

Attachment Number 001 

Attachment Name 

19976 904 Protocol-19976-904 PR redacted 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

EPAX120915024296082 
Multigeneration Reproduction Study in Rats 

Work Request Number 

Service Code 

Protocol 

Performing Laboratory: 
Nlultigeneration Reproduction Study in Rats 

TABLE OF CONTENTS 

Pagc 

TABLE OF CONTENTS .................................................................................................................2 

INTRODUCTION...........................................................................................................................3 

SPONSOR AND CONTACT INFORMATION .............................................................................4 

REGULATORY COMPLIANCE AND TEST GUIDELINES ......................................................4 

ANIMAL WELFARE ACT COMPLIANCE ..................................................................................5 

STUDYDESIGN .............................................................................................................................6 

A. Treatment Groups and Exposure Concentrations ................................................................6 

B. Treatment Schedule .............................................................................................................6 

C. Sacrifice Schedule ................................................................................................................6 

MATERIALSAND METHODS .....................................................................................................7 

A. Test Substance .....................................................................................................................7 

B. Test System ..........................................................................................................................7 

C. Animal Husbandry ...............................................................................................................7 

D. Acclimation Period ..............................................................................................................9 

E. Randomization ...................................................................................................................10 

F. Duration of Exposure .........................................................................................................10 

G. Inhalation Exposure System ..............................................................................................10 

H. Characterization of Chamber Atmosphere .........................................................................1 l 

I. In-Life Observations - Parental Animals ...........................................................................12 

J. Breeding .............................................................................................................................13 

K. Estrous Cycle Evaluation ...................................................................................................14 

L. Gestation Procedures .........................................................................................................14 

M. Lactation Procedures (Fi and F2 Offspring) .......................................................................14 

N. Developmental Landmarks - F, Generation ......................................................................15 

O. FZ Offspring .......................................................................................................................15 

P. Anatomic Pathology Evaluation ........................................................................................15 

Q. Sperm Assessment .............................................................................................................18 

STATISTICALANALYSES ........................................................................................................18 

A. Reproductive Function Calculations ..................................................................................18 

B. Statistical Methods .............................................................................................................19 

SAFETY AND HOUSEKEEPING ...............................................................................................20 

RECORDS AND SAMPLE STORAGE .......................................................................................20 

STUDYPERSONNEL ..................................................................................................................20 

STUDYDATES ............................................................................................................................20 

REFERENCES ..............................................................................................................................20 

SIGNATURES..................................................................................................................... ..........22 

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ylultigeticration Reproduction Study in Rats 

INTRODUCTION 

The test substance, is a volatilc fluorocarbon undcrgoing cvaluation for rcgistration in 

various global regions. 

A developmental study was conducted in time-mated pregnant female rats that were exposed 

whole body to concentrations of 0, 500, 1500, or 10,000 ppm, 6 hours per day for gestation days 

6 through 20.(3)Test substance-related, adverse decreases in maternal body weight and weight 

gain were observed in maternai rats exposed to 10,000 ppm. Body weight of 10,000 ppm 

maternal animals (corrected for the weight of the gravid uterus) was 12% lower compared to the 

controls. Cumulative maternal body weight gain for the 10,000 ppm group was 38% lower 

compared to the control values, and net body weight gain (corrected for the products of 

conception) was 91% lower than control. In addition, a test substance-related increase in the 

incidence of pallor was observed in 10,000 ppm maternal rats. Test substance-related, decreases 

in fetal body weight were observed in offspring from rats exposed to 10,000 ppm (16% lower 

compared to control). There were no test substance-related effects on the incidence of fetal 

malformations or variations at any exposure concentration. The NOAEL for maternal and fetal 

effects was 1500 ppm based on decreased maternal and fetal body weight at 10,000 ppm. 

In a previous 90-day inhalation study, male and female rats were exposed whole body for 

6 hours per day, 5 days per week to 0, 510, 1500, or 10,000 ppm of the test substance over a 

90-day period. (')Test substance-related decreases in body weight, food consumption, and food 

efficiency were observed in males and females exposed to 10,000 ppm. Clinical signs of toxicity 

and neurobehavioral changes that were considered to be secondary to the systemic toxicity 

observed in 10,000 ppm males included abnormal gait (1 of 20 animals), aggressive behavior 

(1 of 20 animals), ear twitching (1 of 20 animals), hyperactivity (1 of 20 animals), low posture 

(1 of 20 animals), tremors (1 of 20 animals), hyper-reactivity (3 of 20 animals), decreased 

hindlimb grip strength, hyper-reactivity during handling (3 of 20 animals), high arousal (3 of 

20 animals), and slight (not statistically significant) decreases in motor activity. There were no 

test substance-related clinical signs of toxicity or neurobehavioral changes in females at any 

exposure concentration. There were no adverse, test substance-related effects on hematology, 

clinical chemistry, or organ weight parameters in males or females exposed to any concentration 

of the test substance. Minimal, non-adverse, morphological changes in the anterior nose, femur, 

and/or teeth were observed in males at 510 ppm and above. The No-Adverse-Effect Level 

(NOAEL) for this study was considered to be 1500 ppm in male and female rats based on test 

substance-related reductions in body weights in males and females exposed to 10,000 ppm. 

A second 90-day inbalation study was conducted to further refine the NOAEL, and to determine 

the potential for the test substance to cause DNA damage. In this study male and female rats 

were exposed whole body for 6 hours per day, 5 days per week to 0, 3000, 4000, 5000, or 

7500 ppm over a 90-day period. (2)Test substance-related, adverse effects on body weight and 

food consumption were observed in male rats exposed to 7500 ppm. No test substance-related 

changes in body weight, food consumption, or food efficiency were observed in males exposed 

to concentrations up to and including 5000 ppm or in females exposed to any concentration. No 

test substance-related adverse clinical signs of toxicity, effects on ophthalmology parameters, 

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Multigeneratiorn Reproduction Study in Rats 

number of micronucleated polychromatic erythrocytes, clinical pathology parameters, organ 

weight parameters or gross morphological changes were observed in males or females exposed 

to any concentration. Test substance-related, non-adverse microscopic findings were observed in 

teeth and femur/knee joints of all male and female rats exposed to the test substance. The 

NOAEL for male rats was considered to be 5000 ppm based on decreased body weight and food 

consumption at 7500 ppm. The NOAEL for female rats was considered to be 7500 ppm, the 

highest concentration tested. 

The proposed exposure concentrations for the current multigeneration reproduction study are 0, 

1500, 5000, and 8500 ppm based on the decreased body weight gain observed in males at 

7500 ppm and above, and on the decreased body weight and/or weight gain in non-pregnant 

females, gestating females, and fetuses observed at 10,000 ppm. 

The objective of this study is to evaluate the effect of the test substance on the gonadal function, 

conception, parturition, and growth and development of male and female Cr1:CD(SD) rats over 

2 generations involving the production of at least one set of litters in each generation. The 

inhalation route of exposure was chosen because it is the potential route for human exposure. 

Air-exposed groups of male and female rats will serve as negative controls. 

SPONSOR AND CONTACT iNFORMATION 

Sponsor: 

Sponsor Contact: 

Testing Facility Contact: 

Sponsor Approval: found on the Work Authorization Form 

REGULATORY COMPLIANCE AND TEST GUIDELINES 

Except as noted below, this study will be conducted in compliance with the following good 

laboratory practice(s), which are compatible with current OECD Good Laboratory Practices: 

• U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards 

— The test substance will be characterized by the sponsor prior to the initiation of this study. 

Although the characterization will not be performed under Good Laboratory Practice 

Standards, the accuracy of the data is considered sufficient for the purposes of this study. 

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Multigeneration Reproduction Study in Rats 

This study will be conducted in compliance with the following test guideline(s): 

• U.S. EPA, OPPTS 870.3800: Reproduction and Fertility Effects, Health Effects Test 

Guidelines (1998) 

• OECD, Section 4(Part 416): Two-Generation Reproduction Toxicity Study, Guideline for 

the Testing of Chemicals (2001) 

ANIMAL WELFARE ACT COMPLIANCE 

This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act 

regulations (9 CFR) and the Guidelines from the Guide for the Care and Use of Laboratory 

Animals (NRC 2011). All studies conducted by or for 

( adhere to the following principles: 

• The sponsor and/or the study director ensure that the study described in this protocol does not 

unnecessarily duplicate previous experiments, and is in compliance with the Policy 

on Animal Testing. 

• Whenever possible, procedures used in this study have been designed to implement a 

reduction, replacement, and/or refinement in the use of animals in an effort to avoid or 

minimize discomfort, distress or pain to animals. All methods are described in this study 

protocol or in written laboratory standard operating procedures. 

• policy is that animals experiencing severe pain or distress that cannot be 

relieved are painlessly euthanized, as deemed appropriate by the veterinary staff and study 

director or appropriate designee. The sponsor will be advised by the study director of all 

circumstances that could lead to this action in as timely a manner as possible. 

• Methods of euthanasia used during this study are in conformance with the above referenced 

regulation and the recommendations of the American Veterinary Medical Association 

(AVMA 2007) Guidelines on Euthanasia. 

• Animals will be provided with species-appropriate environmental enrichment. 

• The procedures in this protocol have been reviewed by the 

and comply with acceptable standards for animal welfare and humane care. 

• is accredited by the Association for the Assessment and Accreditation of 

Laboratory Animal Care (AAALAC) Intemational. 

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Multigeneration Reproduction Study in Rats 

STUDY DESIGN 

A. Treatment Groups and Exposure Concentrations 

Group Number/ Targeted Atmospheric 

Male Female Group Concentration (ppm) 

1 1 30 0 (Control) 

2 2 30 1500 

3 3 30 5000 

4 4 30 8500 

B. Treatment Schedule 

Approximate 

Approximate Age at Start of Number of Study 

Test Substance Administration Days Before Duration of Test Substance 

Generation (days) Mating Administration 

Pt 56 70 (at least) Until the day prior to sacrifice 

Ft 21 70 (at least) Until the day prior to sacrifice 

Study Phase Duration of Test Substance Exposure 

Premating period (PI, F 1 ) At least 10 weeks 

Mating period (Pl , F I ) Up to 2 weeks 

Postmating period (PI, Fi) 

Males 5-6 weeks after the end of the mating period 

Females with no evidence of mating or no At least 24 days after the end of the mating 

litter delivered period 

Gestation period (PI, F 1 ) Approximately 20 days (GD 0-19) 

Lactation period (P l , FI ) Approximately 18 days (LD 7-21) 

F2Offspring No direct exposure 

C. Sacrifice Schedule 

Animals Generation Schedule 

Adult Males Pi, Fl After siring litters (at discretion of study director) 

Pregnant Females P1, F, On day of weaning litters (day 21 postpartum) 

Non-pregnant Females Pi, Fi Approximately day 24 after the end of cohabitation 

Culled Pups F1 , F2 Day 4 postpartum 

Weanlings/Offspring Fl On day of weaning (day 21 postpartum, except F I 

animals selected for continued evaluation; see section 

E.2) 

Offspring F2 Post-natal day 21 

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Multigcncration Reproduction Study in Rats 

MATERIALS AND METIIODS 

A. Test Substance 

1. Identification 

The test substance, will be supplied by the sponsor. The test batch used for this study 

was assigned number . Available information on the purity, composition, 

contaminants, synonyms, CAS registry number, basic physical properties, hazards, and 

hazardous material classification(s) will be provided by the sponsor. The sponsor reported purity 

is o%. 

2. Test Substance Stability 

The stability of the test substance over the course of the study will be confirmed by analysis of 

chamber concentration over the course of the study. 

B. Test System 

Approximately 130 male and 130 female (nulliparous and non-pregnant) Cr1:CD(SD) rats (no 

male-female siblings), requested to be approximately 42 days of age, and weighing 

approximately 201 to 225 grams and 176 to 200 grams, respectively, will be obtained from 

Charles River Laboratories Intemational, Inc., Raleigh, North Carolina. The weight range is not 

a factor for the purposes of this study and, therefore, deviations outside of this range are expected 

not to have any impact on the study. The body weight range will be documented in the study 

records and provided in the final report. 

The rat was selected for this study because it is a preferred species for reproductive toxicity 

testing and recommended by regulatory guidelines. The Cr1:CD(SD) strain was chosen because 

extensive background data are available from the literature, the supplier, and previous studies at 

This strain is also considered suitable relative to longevity, hardiness, and 

incidence of spontaneous disease. 

C. Animal Husbandry 

Housing During Non-Exposure Periods 

All animals will be housed individually during non-mating periods in solid-bottom caging with 

bedding containing acceptable species-specific enrichment. 

a. Pretest and Premating 

All male and female animals will be housed individually in solid-bottom caging with bedding 

containing acceptable species-specific enrichment. Each cage rack will contain only animals of 

one sex. 

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Multigeneration Reproduction Study in Rats 

b. Cohabitation Period 

All animals will be housed as breeding pairs (female placed in the male's cage) in solid-bottom 

caging with bedding containing acceptable species-specific enrichment. Twenty days affter the 

first day of cohabitation, females without evidence of copulation will be housed singly in solidbottom caging with bedding containing acceptable species-specific enrichment. 

Gestation Period 

Days 0-Delivery: Females assumed to be pregnant will be housed individually in solid-bottom 

caging with bedding containing acceptable species-specific enrichment. Females assumed not 

pregnant will be housed in the same manner on the day that mating pairs are separated. 

d. Lactation Period 

Adult females will be housed with their litters in solid-bottom caging with bcdding corltaining 

acceptable species-specific enrichment 

2. Environmental Conditions During Non-Exposure Periods 

Animal rooms will be maintained at a temperature of 20-26°C (68-79°F) and a relative humidity 

of 30-70%. Animal rooms will be artificially illuminated (fluorescent light) on an approximate 

12-hour light/dark cycle. The temperature and relative humidity values in the housing rooms are 

monitored continuously and recorded automatically at regular intervals each day. The 

temperature and relative humidity records are reviewed by the laboratory veterinarian and/or 

designee. In addition, temperature and relative humidity will be recorded during mortality and 

moribundity checks, and retained with the study records. Unless judged by the study director or 

the laboratory veterinarian to have significantly affected the results of the study, the temperature 

and relative humidity ranges in the housing rooms will not be included in the fmal report. 

3. Feed and Water 

Except during exposures, PMI ®Nutrition International, LLC Certified Rodent LabDiet ~ 5002 

and tap water will be available ad libftum. 

4. Identification 

a. Pretest (PI) 

Temporary pretest number recorded on the cage label. 

b. After Assignment to Treatment Groups (Pi and F, Adults) 

Each parental animal will be assigned a permanent animal number at grouping (Pi), or on postnatal day 21 for F, adults designated for continuation on study. This number will be recorded on 

the cage label and tattooed on the tail. 

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Multigeneration Reproduction Study in Rats 

5. Animal Health and Environmental Monitoring Program 

As specified in the animal health and environmental monitoring program, the 

following procedures are performed periodically to ensure that contaminant levels are below 

those that would be expected to impact the scientific integrity of the study: 

• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, 

and other contaminants. 

• Samples from freshly washed cages and cage racks are analyzed to ensure adequate 

sanitation by the cagewashers. 

Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional 

requirements and not to exceed stated maximum concentrations of key contaminants, including 

specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The 

presence of these contaminants below the maximum concentration stated by the manufacturer 

would not be expected to impact the integrity of the study. 

The animal health and environmental monitoring program is administered by the attending 

laboratory animal veterinarian. Data are maintained separately from study records and may be 

included in the final report at the discretion of the study director. 

D. Acclimation Period 

Upon arrival at all animals will be housed in quarantine. The animals will be: 

• quarantined for at least 5 days. 

• individually housed in polycarbonate solid-bottom caging with bedding containing 

acceptable species-specific enrichment. 

• identified temporarily by a pretest number recorded on the cage label. 

• weighed during acclimation. 

• observed with respect to weight gain and any gross signs of disease or injury. 

The animals will be released from quarantine by the animal resources supervisor or designee 

based on body weights and clinical signs. 

Animals that are accidentally killed or removed from study during the acclimation period will be 

discarded without necropsy. Animals that are found dead or euthanized during the acclimation 

period will be given a gross examination to check for the presence of disease. Dependent upon 

these findings, further diagnostic procedures may be employed at the discretion of the study 

director, a pathologist, or the laboratory animal veterinarian. The results will not be reported in 

the final report unless considered significant to the evaluation of the study. 

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Multigeneration Reproduction Stuciy in Rats 

E. Randomization 

1. PI Generation 

Animals of each sex will be ranked by their most recently recorded body weight and randomly 

assigned to control and experimental groups using a computerized randomization procedure 

designed to produce a homogeneous distribution of body weights across groups. The 

randomization should result in a distribution in which the mean body weights for all groups 

within a sex are not statistically different (p > 0.05). Animals that lose excessive weight or that 

are ill prior to the start of treatment will be removed from the study and replaced, if possible. 

Animals that have not been assigned to a test group, or that are removed from study on test day 0 

for out-of-range body weights, will be released for other laboratory purposes, or be sacrificed by 

carbon dioxide asphyxiation while under isoflurane anesthesia and discarded without anatomic 

pathology evaluation, at the discretion of the study director. 

2. F, Generation 

On day 21 postpartum, offspring in the F tlitters of each treatment level will be randomly 

selected (one animal/sex/litter, when possible) to serve as parents for the F 2generation. For 

groups without sufficient litters and at the discretion of the study director, additional pups will be 

chosen from randomly selected litters within the group to achieve the required group size. 

Selection of animals within litters will be random. 

F. Duration of Exposure 

In general, each group of P, animals will be exposed for 6 hours/day, 7 days/week according to 

the schedule shown in Table B. Beginning on post-natal day 21, F, offspring selected for the 

parental generation will be exposed for 6 hours per day, 7 days/week according to the schedule 

shown in Table B. Exposure of gestating and lactating females will not be conducted during 

gestation days 20-delivery, and lactation days 0-6 for compliance with animal welfare 

considerations. F2 offspring will not be directly exposed to the test substance. 

The starting time of each exposure will be defined as the time when the generation system is 

turned on. The ending time of each exposure will be defined as the time when the generation 

system is turned off. Animals will be exposed to the test substance during both the time it takes 

for the chamber to reach concentration and the time it takes for the test substance to be purged 

from the chamber. After each exposure, animals will be retumed to their cages. The exposure 

period is defined as the period between initiation of the first exposure and completion of the last 

exposure. 

G. Inhalation Exposure System 

1. Atinosphere Generation 

Chamber atmospheres will be generated by flash evaporation of the liquid test substance in air 

with a round-bottom, flash evaporation flask. The test substance will be metered into the 

evaporation flask with a pump. The evaporation flask will be heated to vaporize the test 

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Multigeneration Reproduction Study in Rats 

substance. Houseline generation air, metered to the evaporation flask by a mass flow controller, 

will carry the vaporized test substance into a dilution air stream leading to the exposure chamber. 

Chamber concentrations of test substance will be controlled by varying the test substance feed 

rate to the heated flask or by varying the generation/dilution air supply to the exposure chambers. 

Test atmospheres will be exhausted into the exhaust stack. 

All components of the test substance generation system will be fabricated from stainless steel, 

glass, Neoprene®, Teflon®, or other non-reactive materials. The actual generation equipment and 

conditions used for test substance generation will be documented in the study records and final 

report. 

2. Chamber Construction and Design 

Exposure chambers will be constructed of stainless steel and glass (NYU style) with a nominal 

internal volume of 750 L(test chambers) and 1000 L(control chamber). A tangential feed inside 

the chamber will promote uniform chamber distribution of the test atmosphere. The chamber 

volume will be chosen so that the total body volume of the test animals does not exceed 5% of 

the chamber volume. 

3. Exposure Mode 

During exposure, animals will be individually placed in stainless steel, wire-mesh modules and 

exposed, whole-body, inside the exposure chamber, except during the cohabitation period, when 

animals will be housed as mating pairs and exposed in the same manner. The modules will be 

placed on a stainless steel stand inside the exposure chamber so that the animals will be elevated 

from the bottom of the exposure chamber. 

H. Characterization of Chamber Atmosphere 

1. Test Substance Sampling and Analysis 

During each exposure, the atmospheric concentration of the test substance will be detenmined by 

gas chromatography at approximately 60-minute intervals in the test chambers. The control 

chamber will be monitored at least once per week. Known volumes of chamber atmosphere will 

be continually drawn from the chamber and will be directly injected into a gas chromatograph 

equipped with an automated gas sample valve and a flame ionization detector. The column used 

and the conditions of the chromatograph will be documented in the study records and identified 

in the final report. The atmospheric concentration of the test substance will be determined from 

a standard curve derived from vapor standards. Standards will be prepared prior to the exposures 

by injecting known volumes of the liquid test substance into Tedlar ®bags containing known 

volumes of air. 

Upon completion of the exposures, Camile Inhalation Toxicology Automated Data System 

(CITADS) sample results will be transferred to the Camile Inhalation Reporting and Analysis 

System (CIRAS), which collates sample calculations. 

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ViuitiQerieration Reprodtiction Studv in 12ats 

2. Environmental Monitoring 

Chamber temperature will be targeted at 19-25°C and recorded approximately every hour during 

each exposure. Chamber relative humidity will be targeted at 30-70% and recorded 

approximately every hour during each exposure. Temperature and humidity will be measured 

with an appropriate thermo-hygrometer. Chamber airflow will be set at the beginning of each 

exposure to achieve at least 10 air changes per hour, monitored continually with appropriate 

equipment, and recorded initially, approximately every hour, and whenever changes are made 

during each exposure. Chamber oxygen concentration will be targeted to be at least 19%, 

measured with an appropriate oxygen analyzer and recorded once during each exposure. 

Supplemental oxygen will be added to the chamber atmospheres, if needed, to maintain the target 

oxygen concentration. In that case, chamber oxygen concentration will be measured and 

recorded 3 times during each exposure. 

Chamber Distribution of Test Substance 

The distribution of the test substance will be determined in the high-concentration chamber. 

I. In-Life Observations - Parental Animals 

1. ClinicalObservations 

Cage-site examinations will be conducted at least once daily throughout the study. Moribund 

animals will be sacrificed. At least once weekly throughout the premating treatment, gestation, 

and lactation periods, each of the P, and F tparental animals will be individually handled and 

carefully examined for abnormal behavior and/or appearance. 

2. Body Weight Measurements 

Females without evidence of copulation, those that copulate and do not deliver a litter, aiid malcs 

will be weighed on a weekly schedule and at sacrifice. 

a. Premating and Cohabitation Periods 

All P, and F, animals will be weighed once a week. All animals being evaluated for 

developmental landmarks (vaginal patency and preputial separation) will be weighed the day of 

achievement. 

b. Gestation and Lactation Periods 

P, and F, females will be weighed on days 0, 7, 14, and 20 of gestation and on days 0, 7, 14, and 

21 of lactation. 

3. Food Consumption and Food Efficiency 

During the test period, the amount of food consumed by each animal over the weighing interval 

will be determined by weighing each feeder at the beginning and end of the interval and 

subtracting the final weight of the feeder during the interval from the initial weight. From these 

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Multigeneration Reproduction Study in Rats 

measurements, mean daily food consumption over the interval will be determined. From the 

food consumption and body weight data, the mean daily food efficiency will be calculated. Feed 

spillage will not be recorded. However, excessive spillage will be documented in the study 

records. 

a. Premating and Cohabitation Pcriods 

Individual food consumption will be determined weekly for all P, and F, animals throughout the 

premating period ending approximately at test day 70. From the food consumption and body 

weight data, the mean daily food efficiency will be calculated. Food consumption will not be 

measured during cohabitation. After mating pairs have been separated, food consumption will 

not be measured in males or in females with no evidence of copulation. 

b. Gestation and Lactation Periods 

Individual food consumption of pregnant P, and F, females will be recorded on gestation days 0, 

7, 14, and 20 and on lactation days 0, 7, and 14. From the food consumption and body weight 

data, the mean daily food efficiency will be calculated. 

J. Breeding 

Start of Cohabitation 

Animals will be co-housed for mating after approximately 10 weeks of exposure to the test 

substance. The day animals are first co-housed is designated as day 1 of cohabitation. Each 

female will be continually housed on a 1:1 basis with a randomly selected, non-sibling male of 

the same exposure level, in the male's cage or exposure module. 

Based on the outcome of the first mating and at the discretion of the study director, the females 

may be cohoused again in the manner outlined above, except with different males, to produce a 

second set of litters (F]b or F20. 

A second mating may be conducted when poor reproductive performance is observed in the 

control group or in the event of equivocal treatment-related reduction in reproductive 

performance. If production of a second litter is deemed necessary in either generation, the dams 

should be remated approximately 1 to 2 weeks following weaning of the last Fl aor F2alitter. 

2. Duration of Cohabitation Period 

Animals will be co-housed until evidence of copulation is observed or until2 weeks elapses. On 

the day copulation is confirmed, the female will be transferred back to individual cage housing. 

The cohabitation period will end on the morning of day 14 of cohabitation. 

3. Evidence of Copulation 

Once daily, each female will be examined for the presence of an intravaginal copulation plug or 

sperm in vaginal lavage sample, either of which will be considered evidence of copulation. The 

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Multigeneration Reproduction Study in Rats 

presence of an intravaginal plug and/or sperm will be recorded. The day evidence of copulation 

is observed is designated as day 0 of gestation (GD 0). 

K. Estrous Cycle Evaluation 

Vaginal lavage samples will be collected daily from all P, and F, female animals to determine 

the stages of the estrous cycle. Vaginal lavage samples will be collected beginning 3 weeks prior 

to start of cohabitation and continuing until copulation is confirmed or the cohabitation period 

ends. The vaginal lavage sample collected on the day copulation is confirmed will not be used 

for estrous cycle evaluation. Vaginal lavage samples will also be collected from all P, and F, 

parental female animals at the time of sacrifice. Vaginal lavage samples will be examined 

microscopically for determination of the stage of the estrous cycle (diestrus, estrus, or proestrus). 

L. Gestation Procedures 

Beginning on day 20 of gestation for mated females, or at the end of the cohabitation period for 

females without evidence of copulation, female animals will be observed at least twice daily for 

signs of delivery and offspring. 

M. Lactation Procedures (F, and FZOffspring) 

The day when delivery is complete is designated day 0 postpartum. At each examination period 

(days 0, 4, 7, 14, and 21 postpartum), offspring will be individually handled and examined for 

abnormal behavior and appearance; any dead or abnormal pups will be recorded. Dams that 

have no live pups remaining during the lactation period will be sacrificed at the discretion of the 

study director. Litters of dams that die during lactation will be sacrificed. 

1. Day 0 Postpartum 

Live and dead pups in each litter will be counted by sex as soon as possible after delivery is 

completed. Live pups in each litter will be individually weighed. 

2. Day 4 Postpartum 

Pups in each litter will be counted by sex and individually weighed. Litters will be culled 

randomly to 8(4/sex when possible) and the number of pups of each sex recorded. Extra 

offspring will be euthanized (by decapitation) and discarded without anatomic pathology 

examination. Litters of 8 offspring or fewer will not be reduced. 

3. Days 7 and 14 Postpartum 

Pups in each litter will be counted by sex and individually weighed. 

4. Day 21 Postpartum (Weaning - PND 21) 

Pups in each litter will be counted by sex and individually weighed. Offspring in the F, litters of 

each treatment level will be randomly selected (one animal/sex/litter when possible) to serve as 

parents for the F2 generation and will be placed in individual cages. For groups without 

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Multigeneration Reproduction Study in Rats 

sufficient litters and at the discretion of the study director, additional pups will be chosen from 

randomly selected litters within the group to achieve the required group size. Selection of 

animals within litters will be random. 

N. Developmental Landmarks - F, Generation 

1. Vaginal Patency 

F, female animals designated for mating will be examined for vaginal patency once daily 

beginning on post-natal day 21 until achievement or post-natal day 43, whichever comes first. 

Body weight will be recorded on the day of achievement. 

2. Preputial Separation 

F, male animals designated for mating will be examined for preputial separation beginning on 

post-natal day 35 until achievement or post-natal day 55, whichever comes first. Body weight 

will be recorded on the day of achievement. 

O. F2 Offspring 

Anogenital Distance 

Anogenital distance will be recorded in F Zpups on day 0 postpartum, if treatment-related effects 

are observed on sex ratio or sexual maturation of F ipups or at the discretion of the study 

director. 

2. Exposures 

F2 Offspring will not be directly exposed to the test substance, 

P. Anatomic Pathology Evaluation 

The anatomic pathology findings will be peer reviewed by a second veterinary pathologist. 

1. Pt and F, Adults 

a. Method of Euthanasia 

Animals will be sacrificed by isoflurane anesthesia and exsanguination. The order of sacrifice 

for scheduled deaths will be random among all treatment groups within a sex. 

b. Postmortem Examinations 

All animals will receive a gross pathology examination, including those that die (found dead, 

accidentally killed) or are euthanized prior to scheduled sacrifice and those for which copulation 

does not result in production of offspring. The uteri of all cohabited females will be examined 

for the presence and number of implantation sites. All gross lesions will be preserved in 

appropriate fixative for possible future histopathology examination. 

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Mtiltigeneration Reproduction Study in Rats 

C. Organ Weights from P, and F, Animals Euthanized at Scheduled Sacrifice 

The following tissues will be collected from all Pl and Ft parental animals euthanized at 

scheduled sacrifice and weighed. 

Both Sexes Male Female 

liver testes ovaries (including oviducts) 

kidneys epididymidesa uterus (including cervix) 

spleen right cauda epididymis a 

pituitary gland prostate 

thyroid seminal vesicles with coagulating 

adrenal glands glands (including fluids) 

brain 

a Right and left epididymides will be weighed together as a unit. The right cauda epididymis 

will be subsequently removed and weighed, and used for sperm motiliry and morphology. The 

left epididymis and left cauda epididymis will be frozen in liquid nitrogen and used for sperm 

counts. The left cauda epididymis will be weighed prior to spertn count evaluation. 

Relative organ weights (percent of terminal body weight; percent of brain weight) for weighed 

organs will be calculated. Male accessory sex gland weights will be calculated by addition of the 

prostate and seminal vesicles with coagulating gland (including fluids). Terminal body weights 

determined just prior to necropsy will be used in the assessment of organ weight changes. 

d. Tissues Collected from P, and F1 Animals Euthanized at Scheduled 

Sacrifice 

The following tissues will be collected from all P tand Ftanimals euthanized at scheduled or 

unscheduled sacrifice, and in animals found dead or accidentally killed. 

Both Sexes Male Female 

pituitary gland testis ovaries 

adrenal glands epididymisb oviducts 

gross observations a prostate uterus 

seminal vesicles cervix 

coagulating glands vagina 

a Gross observations made at necropsy for which 

histopathology is not appropriate (e.g., fluid, ruffled fur, 

and missing anatomic parts) will not be collected. 

b The right testis and right epididymis will be placed in 

modified Davidson's solution and processed for 

histopathology. The left testis and left epididymis will be 

frozen for sperm assessment (see section Q) and will not be 

processed for histopathology. All other tissues 

(reproductive and non-reproductive) collected from male 

and female animals will be placed in formalin. 

Histopathology examination of these tissues will be conducted for the first 10 parental animals 

(the first 10 consecutive animal numbers in each group) surviving to scheduled sacrifice in the P t 

-16- 
Multigeneration Reproduction Study in Rats 

and F, male and female high-treatment and control groups. Organs demonstrating treatmentrelated changes will be examined for the remainder of the high-treatment and control animals 

and for all parental animals in the mid- and low-treatment groups to further delineate or establish 

a"no-observed-adverse-effect level". In addition, reproductive organs in animals with suspected 

impaired reproductive performance (e.g., failure to mate, conceive, sire, or deliver healthy 

offspring; or effects in estrous cyclicity or sperm parameters) will be evaluated from all groups. 

In addition, a quantitative evaluation of primordial and growing follicles will be conducted on 

the first lO lactating F, females (surviving to scheduled sacrifice) from control and hightreatment groups. If a treatmerit-related change is observed, other treatment groups (10 lactating 

F, females per group) will be examined to further delineate or establish a "no-observed-adverseeffect level". Six ovarian cross sections (5 μm thick) will be taken from the central area of each 

ovary using a step section technique. Primordial and growing follicles (up to but not including 

antral follicles) will be enumerated for up to 12 ovarian sections per animal. 

Most gross lesions from low and intermediate levels will also be evaluated microscopically. 

Selected gross observations for which a microscopic diagnosis would not be additive 

(e.g., pododermatitis, tail chronic dermatitis, calculus, and deformities of the toe, tail, or ear 

pinnae) will be saved, but will not be processed for microscopic evaluation. 

Gross lesions from animals that are found dead or accidentally killed (tissue integrity 

permitting), or are euthanized prior to scheduled sacrifice may be further processed to slides for 

possible histopathology evaluation at the discretion of the study director or pathologist. 

Tissues from animals removed from study will not be processed for microscopic evaluation 

unless considered necessary by the study director or pathologist. 

2. Offspring 

a. Method of Euthanasia 

Animals will be sacrificed by decapitation (for pups up to lactation day 4) or euthanized by an 

intraperitoneal injection of a commercial euthanasia agent (lactation days 5-20). 

b. Postmortem Examinations 

Offspring that are moribund, found dead, or sacrificed because of death of the dam during the 

lactation period will undergo gross pathology evaluation. Dead pups will be examined to the 

extent possible. 

3. F, and F2 Weanlings 

a. Method of Euthanasia 

Animals will be sacrificed by isoflurane anesthesia and exsanguination. 

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Multigeneratiori Reproductiorl Study in Rats 

b. Postmortem Examinations 

Three weanlings/sex/litter (litter size permitting) will undergo gross pathology evaluation. All 

gross lesions and target organs will be preserved for possible future histopathology examination. 

One weanling/sex/litter will be designated for organ weight and histopathology evaluation. 

These weanlings will have brain, spleen, and thymus weights recorded and all gross lesions and 

target organs will be preserved in formalin for possible future histopathology examination. 

Microscopic evaluation of the preserved gross lesions will be conducted at the discretion of the 

study director or pathologist. 

Q. Sperm Assessment 

Sperm parameters for all Pt and F, parental males will be evaluated. The right epididymis will 

be removed, and the right cauda epididymis will be weighed. Sperm will be collected from the 

right cauda epididymis for evaluation of motility and morphology. The left epididymis and testis 

will be frozen in liquid nitrogen and stored between -65°C and -85°C for sperm and spermatid 

counts, respectively. 

STATISTICAL ANALYSES 

A. Reproductive Function Calculations 

The following table lists the indices of reproductive function that will be calculated for the P, 

and Ft parental animals. 

number copulateda Mating Index (%) = x 100 

nun~ ber cohabited 

Fertility Index (%) = number pregnant a 

x 100 number copulated 

Gestation Index (%} = number of litters with at least one live pup x 100 number of litters 

number of implantation sites - number of pups born Post-Implantation Loss (%)` = x 100 number of implantation sites 

Pups Born Alive (%)` = number of pups born alive x 100 number of pups born 

Viability Index (%)`° d= 

number ofpups alive day 4 preculling x 100 number of pups born alive 

Lactation Index (%)`' d = number of pups alive at weaning (Day 21 postpartum) x 100 

number of pups alive day 4 postculling 

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Multigeneration Reproduction Study in Rats 

Litter Suwival(%)d= 

number of litters weaned 

number of viable litters delivered x 100 

a Evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, found dead 

pregnant, or delivery of a litter. 

b Including those found dead pregnant during gestation. 

c Determined for each litter. Mean and standard deviation for each treatment level will be 

calculated. 

d Excluding litters sacrificed due to death of dam during lactation. 

B. Statistical Methods 

Significance will be judged at p< 0.05. Separate analyses will be performed on the parental data 

collected for each sex. Other methods will be used, if appropriate, at the time of analysis. The 

statistical methods used will be described in the final report. 

Method of Statistical Anal sis 

If preliminary test is not If preliminary test is 

Parameter PreliminaryTest significant significant 

Body Weight 

Body Weight Gain 

Food Consumption 

Food Efficiency Transforms of the data to 

Gestation Length achieve normality and 

Implantation Site Numbers variance homogeneity will 

Number of Pups per Litter Levene's test for One-way analysis of be used. The order of 

Precoital Interval homogeneity ~ a> and 

vari ance tb~ followed by transforms attempted will 

Vaginal Patency Shapiro-Wilk test (

5) for ~ ~ett's testt't be lo s uare-root' and 

g' q Preputial Separation normality rank-order. If the log and 

Anogenital Distance square-root transforms 

Estrous Cycle Parameters fail, the rank-order will be 

Sperm Parameters used. 

Ovarian Follicle Counts 

Mean Pup Weight 

Or an Wei ht 

Viability Index (%) Transforms of the data as an aresine square roo ~ of Live Bom Index (%) 

o None percent/100, then one-way analysis of variance Post-Implantation Loss (/o) , (7) followed by Dunnett s test Sex Ratio % 

Mating Index (%) 

Fertility Index (%) Fisher's Exact test with Bonferroni-Holm 

Gestation Index (%) None adjustmentt")for the number of comparisons to the 

Lactation Index (%) control 

Litter Survival % 

For each parameter analyzed with a trend test, the test will be applied to the data sequentially. If 

a significant treatment-response is detected, data from the top treatment group will be excluded 

and the test repeated until no significant trend is detected. (10)For litter parameters, the 

proportion of affected fetuses per litter or the litter mean will be used as the experimental unit for 

statistical evaluation. ("

) 

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Multigeneration Reproduction Study in Rats 

SAFETY AND HOUSEKEEPING 

Good housekeeping procedures will be practiced to avoid contamination of facilities and 

potential health hazards. To avoid skin contact, gloves will be worn when handling the test 

substance. In addition, the test substance will be handled in properly ventilated areas. Animal 

carcasses and feces will be incinerated. 

RECORDS AND SAMPLE S"I'ORAGE 

All raw data, the protocol, anlendmcnts (if any), and the final report will bc retained. 

STUDY PERSONNEL 

Study Director: 

Primary Technician: <> 

~ 

STiJDY DATES 

Proposcd Experimcntal Start: February 2013 

In-Lifc Complction: Approximately Octobcr 2013 

REFERENCES 

1. (2010). 90-Day Inhalation Toxicity Study in Rats. Unpublished 

rcport, 1 

2. (2011). 90-Day Inhalation Toxicity Study in Rats. IJnpublished 

rcport, 1 

3. (2010). Inhalation Main Developmental Toxicity Study in Rats. 

Unpublished report, 

4. Levene, H. (1960). Robust test for equality of variances. Contributfons to Probability and 

Statistics (J. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto. 

5. Shapiro, S.S. and Wilk, M.B. (1965). An analysis of variance test for nonmality (complete 

samples). Biometrika 52, 591-611. 

-20- 
Multigeneration Reproduction Study in Rats 

6. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6`hedition, pp 246-248 and 

349-352. The Iowa State University Press, Ames. 

7. Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrfcs 20, 

482-491. 

8. Fisher, R.A. (1985). Statistical Methods for Research Workers, 13` hedition. Haffner, 

New York. 

9. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scaradinavian 

Journal of Statistics 6, 65-70. 

10.Selwyn, M.R. (1995). The use of trend tests to determine a no-observable-effect level in 

animal safety studies. Journal of the American College of Toxicology 14(2), 158-168. 

11.Haseman, J.K. and Hogan, M.D. (1975). Selection of the experimental unit in teratology 

studies. Teratology,l2, 165-171. 

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Multigeneration Reproduction Study in Rats 

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