DuPont Chemicals & Fluoroproducts Chestnut Run Plaza 702 1276-E 4417 Lancaster Pike Wilmington, DE 19805 USA 

# aupoNT m 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 CompanySANITIZED 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 EPAX120915024296082Multigeneration 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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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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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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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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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 P t 56 70 (at least) Until the day prior to sacrifice F t 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 (P l , F I ) Up to 2 weeks Postmating period (PI, F i ) 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 , F I ) Approximately 18 days (LD 7-21) F 2 Offspring No direct exposure

C. Sacrifice Schedule
Animals Generation Schedule Adult Males Pi, F l 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 F 1

, 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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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.

-7-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 thefirst 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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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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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 t litters of each treatment level will be randomly selected (one animal/sex/litter, when possible) to serve as parents for the F 2 generation. 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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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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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 t parental 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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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 a or F2 a litter.
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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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 F Z Offspring) 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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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 Z pups on day 0 postpartum, if treatment-related effects are observed on sex ratio or sexual maturation of F i pups 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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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 epididymides a 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 t and F t animals euthanized at scheduled or unscheduled sacrifice, and in animals found dead or accidentally killed.

Both Sexes Male Female pituitary gland testis ovaries adrenal glands epididymis b 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

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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 onthe 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 lactatingF, 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.

-17-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 copulated a

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

-18-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 Inte rv al homogenei ty ~ a> and va ri ance tb ~ followed by transforms a tt empted 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. ( " )

-19-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` h edition, 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` h edition. 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.

-21-Multigeneration Reproduction Study in Rats

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