P.1 

OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

58) ll-J-1 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:16 AM Sheet 58 of 93 
P.2 

R -9D- 01 

TT-T-51 

REPRODUCTIVE TOXICITY TESTING 

by 

CONTINUOUS BREEDING 

f-> 

TEST PROTOCOL IN SWISS (CD-1) MICE 

National Institute of Environmental Health Sciences 

National Toxicology Program 

Developmental and Reproductive Toxicology Group 
P.3 

Jerrold J. Heindel1, J. C. Lamb, IV1*, Robert E. Chapin1, Dushyant K. Gulati2, 

Esther Hope2, Julie George3, C. W. Jameson1, Janet Teague4, and B.A. Schwetz1 

1 National Institute of Environmental Health Sciences 

National Toxicology Program 

P.O. Box 12233 

Research Triangle Park, NC 27709 

2Environmental Health Research and Testing, inc. 

2514 Regency Road 

Lexington, KY 40503 

3 Research Triangle Institute 

P.O. Box 12194 

Research Triangle Park, NC 27709 

4Analytical Sciences, Inc. 

100 Capitola Drive, Suite 1 

Durham, NC 27713 

4 

Present Address: Jellinek, Schwartz, Connolly & Freshman, Inc. 

1350 New York Avenue, N.W., Suite 400 

Washington, DC 20005 

**The procedures described here are current as of January 1989. 

Correspondence to: Dr. Jerrold J. Heindel 

Developmental and Reproductive Toxicology Group 

NIEHS/NTP 

P.O. Box 12233 

Research Triangle Park, NC 27709 
TABLE OF CONTENTS 

P.4 

Topic 

General Study Design 

Methods - General 

Chemical Characterization and Handling 

Chemical/Vehicle Formulations 

Animals 

Statistical 

Methods - Specific 

Task 1 - Dose Range Finding Study 

Task 2 - Continuous Breeding 

Task 3 - Determination of Affected Sex 

Task 3 - Necropsy 

Sperm Motility Determination 

Sperm Count Determination 

Sperm Morphology Evaluation 

Vaginal Cytology Evaluation 

Task 4 - Offspring Assessment 

Results - Summary Data from RACB 

Conclusion 

Flow Diagram of RACB 

Time Line of RACB 

Milestone Chart for RACB 

References 

Pa^e 

3 

5 

6 

8 

11 

13 

17 

17 

19 

23 

25 

26 

27 

28 

28 

32 

35 

41 

Figure 1 

Figure 2 

42 

44 
P.5 

INTRODUCTION 

"Reproductive Assessment by Continuous Breeding (RACB)" is a nev 

standardized protocol designed for routine reproductive toxicity testing. The 

original design vas developed by the National Toxicology Program, Research 

Triangle Park (RTP), NC. The actual testing and standardization vere 

contracted to tvo laboratories: Environmental Health Research & Testing 

(EHRT), Lexington, KY, and Research Triangle Institute (RTI), Research 

Triangle Park, NC. The methods for statistical analysis of the results vere 

provided initially by Program Resources, Inc., RTP, NC, and presently by 

Analytical Sciences, Inc., RTP, NC, under the supervision of the National 

Toxicology Program. 

Until recently, RACB vas generally referred to as "Fertility Assessment 

by Continuous Breeding (FACB)." The title vas revised since the protocol 

provides information on more than fertility. RACB shares the same fundamental 

endpoints as reproductive toxicity protocols recommended by the U.S. 

Environmental Protection Agency (EPA) and the Food and Drug Administration 

(FDA) but is designed to improve the reliability and sensitivity of 

reproductive testing. 

Lamb (1985) provided a comparison of the RACB test system vs. FDA 

segments I, II, and III for drugs, FDA multigeneration studies, and EPA 

tvo-generation studies. Many RACB endpoints (fertility, pups per litter, pup 

veight, sex, and survival) are the same as in conventional EPA and FDA 

protocols. In addition, the RACB protocol tests fertility and reproductive 

outcome under constant challenge. The first generation animals (FQ) are 

housed as breeding pairs continuously for 14 veeks. This approach not only 

increases the number of litters from each breeding pair, but also adds the 
P.6 

dimension of time (progression of an adverse affect) to the evaluation of the 

reproductive performance. Thus, an early effect can be distinguished from a 

later, or progressive, effect. Standard multigeneration studies fail to 

provide such information unless more than one mating per generation is 

performed. 

Furthermore, the statistical pover of the RACB test is considerably 

higher than the conventional studies. The standard multigeneration studies 

require about 20 successful matings per treatment group and at most tvo 

litters per mating pair. The RACB protocol requires 20 males and 20 females 

per treatment group vith 40 control breeding pairs but, because of the 

continuous cohabitation for 14 veeks, as many as 100 litters per treatment 

group can be evaluated. The corresponding number of litters for the control 

group is 175 to 200. 

The purpose of this article is to describe practical details for RACB and 

thereby: 

1) serve as a reference source for reproductive toxicologists and 

regulatory agencies interested in conducting or recommending RACB 

studies, 

2) provide an insight into, and knovledge about, the complexities of the 

RACB test and assist in planning and conducting these studies 

successfully. 

Although RACB can be, in fact has been, adapted for use in rats, the 

majority of the studies have been done in mice. This paper describes the 

standard methodology for conducting the RACB study in mice. 

GENERAL STUDY DESIGN 

The RACB protocol is divided into four tasks (Figures 1 and 2). Task 1 

is a 14-day dose setting study vhich uses five doses and a control (n = 
P.7 

8/sex/group). Endpoints for Task 1 are clinical signs, body veight, and food 

or vater consumption. Task 2 is the continuous breeding phase, consisting of 

a control group (40 breeding pairs) and 3 dose groups (20 pairs/group). Task 

2 dose levels are set so that the highest dose vill be expected to depress 

veight gain by approximately 10% and vill permit >90% survival. The middle 

dose is selected to produce little or no systemic toxicity, vhile the lov dose 

is designed to be a no-effect level. After 7 days of premating exposure to 

the test agent, the animals are housed as breeding pairs for 98 days. 

Endpoints for Task 2 are: clinical signs, parental body veight, fertility 

(number producing a litter/number of breeding pairs), delivery interval, 

litters per pair, live pups per litter, proportion of pups born alive, sex of 

live pups, the pup body veights vithin 18 hr of birth, and food and vater 

consumption. At the end of the 98 days, the pairs are separated and housed 

one animal/cage. Any litters born after the cohabitation phase are reared by 

the dam until weaning (21 days of age). Randomly selected F1 pups are then 

exposed to the test agent at the same concentration as their parents received 

during Task 2. These ?l animals are used for assessment of second generation 

fertility (Task 4, belov). 

Vhen a positive effect on fertility is detected during Task 2, a 1-veek 

crossover mating trial (Task 3) is performed on the parental animals to 

determine vhich sex or sexes may have been affected by the chemical treatment. 

Task 3 is performed after the last Task 2 litter is veaned. The crossover 

mating trial consists of 3 groups of 20 pairs each, control males x control 

females, control males x high-dose females, and control females x high-dose 

males. Pairs are mated for seven days or until a copulatory plug is detected, 

vhichever is first. Animals are not exposed to the test chemical during Task 

3 cohabitation. Dosing is resumed at the appropriate concentrations 
P.8 

after seven days and continued until necropsy. Endpoints for Task 3 are the 

same as Task 2 vith the addition of mating index (number of females plug 

positive/number pairs cohabited). At the end of Task 3, Fo males and females 

are necropsied; the endpoints evaluated are selected organ veight, body 

veight, epididymal sperm motility, morphology, and concentration, and estrous 

cyclicity as monitored by vaginal lavage for the preceding 12 days (some 

studies have used 7 days). Selected organs are examined microscopically after 

fixation in 10% neutral buffered formalin or Bouins fixative and embedding in 

paraffin or plastic; sections are stained vith hematoxylin and counterstained 

vith either eosin or PAS according to standard procedures. 

Task 4, offspring assessment, is conducted vhether or not Task 2 shovs 

reproductive toxicity. In this phase, the last litter from Task 2 is reared, 

veaned, and kept to sexual maturity (74 + 10 days) vhile housed by sex 2 or 3 

per cage, consuming the same concentration of test chemical as their parents. 

At this time, males and females from different litters but vithin the same 

treatment group, are cohabited for seven days until a copulatory plug is 

found, and then housed singly until delivery. The reproductive and fertility 

endpoints for this mating trial are the same as those in Task 3. At the end 

of Task 4, the Fx mice are necropsied; the endpoints are the same as for the 

necropsy of the Fo mice described above. 

METHODS - GENERAL 

All Reproductive Assessment by Continuous Breeding (RACB) studies are 

conducted in accordance vith Good Laboratory Practice (GLP) Regulations 

established by the FDA for Nonclinical Laboratory Studies (Federal Register, 
P.9 

Friday, December 22, 1978, Part II, pp 59986-60025) and vith the requirements 

and recommendations contained in the most recent issue of the "Guide for the 

Care and Use of Laboratory Animals" by Public Health Service, National 

Institutes of Health. 

CHEMICAL CHARACTERIZATION AND SAFETY AND HANDLING PROCEDURES 

The study material obtained for the RACB studies are representative of 

the material to vhich the population at risk are exposed. Most chemicals 

obtained for study in the NTP are unformulated, technical-grade products. 

Formulated products are avoided vhere possible to minimize the number of 

components in the study material vhile still providing an evaluation of the 

chemical (including impurities) to vhich people are most likely to be exposed. 

If, hovever, the chemical is being tested primarily because of an interest in 

its molecular structure or because it is an impurity in a commercial product, 

a pure form of the chemical is obtained because it is more appropriate for 

study. 

Once the study material is procured, a number of chemical analyses and 

methods development are performed. These tasks include both bulk chemical and 

dose formulation analysis. The bulk chemical is analyzed to fully 

characterize the study material prior to use in the RACB program. These 

analyses utilize a vide variety of analytical chemical techniques including 

chromatography, spectroscopy, elemental analysis and physical property 

determinations. These analyses are performed to confirm the identity and to 

determine the purity of the study material. Impurities present in the study 

materiel at levels of IX or greater are routinely identified and quantitated 

to assist in the interpretation of the RACB study. A copy of the chemical 

characterization report is included in the technical protocol as veil as the 

final study report. Procedures for the handling of chemicals are established 
P.10 

to ensure the safety of personnel and the maintenance of the purity and 

stability of the test compound. Information contained in the handling 

protocol specifies safety precautions to be observed, including protective 

equipment to be utilized by personnel. Hazardous characteristics of the 

chemical, such as flammability, explosive properties, and toxicity are listed. 

Materials or substances that are incompatible vith the chemical in question 

are listed so that containers or equipment containing these materials can be 

avoided. Procedures for the decontamination of spills and for the disposal of 

vaste chemical and contaminated materials are also recommended, in addition to 

the first aid procedures. 

Methods for mixing and analyzing dose formulations are developed prior to 

the RACB studies to assure adequate dosing of the study animals. Protocols 

are developed for mixing the study material at the proper dose levels vith the 

appropriate vehicle (feed, vater, corn oil). Homogeneity of feed mixtures and 

other chemical dose formulations that are not solutions is demonstrated before 

the animal study begins. Analysis methods for dose formulations usually 

involves isolation of the study chemical by extraction followed by 

chromatographic or spectroscopic analysis of the extract. 

Stability of both the bulk chemical and dose formulations is also 

determined prior to the RACB studies. Thermally accelerated stability studies 

of the bulk chemical are performed to estimate the long-term stability and to 

aid in setting storage conditions for the chemical. The bulk chemical is then 

routinely monitored for purity during the study. The stability of dose 

formulations for up to three veeks under optimum storage conditions and also 

under simulated dosing conditions is determined. The data obtained from these 

stability studies are used to estimate the shelf life of the dose formulations 

and the conditions under vhich it should be stored to minimize decomposition. 

The dose formulations are routinely analyzed during the RACB studies to insure 

proper doses are administered. 
P.11 

CHEMICAL/VEHICLE FORMULATION 

Chemical/vehicle formulations are all prepared under GLP guidelines. 

Gavage Formulation and Administration - The study chemical is mixed in the 

appropriate vehicle vith concentration determined by the following formula: 

dose level (mg/kg) 

concentration (mg/ml) = 

dose volume (10.0 ml/kg) 

Thus, concentration vill vary vith dose. Each concentration is formulated 

independently on a veight/volume basis in a quantity sufficient to be used 

across a 14-day period of administration if test chemical/vehicle stability 

permits. Aliquots of each formulation of the study chemical and vehicle are 

analyzed prior to onset of exposure for confirmation of dose levels. 

Distilled vater is used for aqueous formulations. The corn oil used as the 

gavage vehicle, is purchased fresh from a local grocer (1 lot/study). Each 

lot of corn oil is monitored for peroxide content every month vhile in use. 

If the peroxide level exceeds 3 meq/kg a fresh lot of corn oil is obtained. 

An aliquot of each formulation of test chemical in vehicle and vehicle are 

archived at -20°C. 

Dosages are adjusted on a veekly basis. Animals are veighed at the 

beginning of the veek and the nev dosage is calculated in relation to the body 

veight. This dosage is used until the animal is veighed again in seven days 

or until delivery of a litter. A dosage volume of 10 ml/Kg body veight (i.e. 

IX body veight) is used. Gavage is accomplished using a lcc Glasspak syringe 

fitted vith an 18/20 guage, 1 1/2 inch curved needle for adult mice or a 20/22 

guage, 1 inch straight needle for the immature mice in Task 4. 
P.12 

Feed Formulation and Dosage Administration - The study chemical is accurately 

veighed and added to a small aliquot of ground feed in a beaker and mixed vith 

a spatula to make a premix. The premix is then added to a preveighed ground 

feed sample and blended in a Patterson-Kelley liquid solids tvin shell blender 

(eight quart model). The blender is run for five minutes, vith the 

intensifier bar on, and for 10 minutes vithout the intensifier (times may be 

varied to obtain homogeniety). Samples from three different places in the 

blender are analyzed from the first mix of a nev study material to assure 

homogeneity of mixing. A different concentration of test chemical is required 

for each dose level. Each concentration is formulated independently in a 

quantity sufficient for a 14-day treatment period if stability of test 

chemical in feed permits. Each formulated feed sample is stored under 

refrigeration prior to use. For each formulation, the quantity prepared is 

based on the assumption that each mouse vill consume at most 10 g per day. An 

aliquot of each formulation of test chemical in feed and the vehicle vill be 

kept for analysis. 

Standard clear glass, amber glass or polycarbonate feeding jars, 

depending on test chemical stability, holding approximately 3.5 oz are used. 

A jar is filled vith the appropriate povdered or granular diet and the veight 

recorded. The filled jar is color coded according to dose group, and placed 

vithin the appropriate cage. 

After seven days, the jar is removed from the cage and reveighed. This 

value is subtracted from the original full veight and the difference is the 

food consumed during that time period. If additional food is required prior 

to the seven-day veighing, a veighed quantity of feed vill be added to the 

feed jar and the amount added vill be noted on the appropriate data sheet. 
P.13 

10 

Determination of average food intake per animal requires division of 

total food consumed in cage by number of animals in the cage. The total food 

intake per animal divided by the number of days vill estimate food intake in 

grams per animal per day. 

Drinking Water Formulation and Administration - The study chemical is 

veighed and mixed vith deionized/filtered vater on a veight to volume basis. 

An aliquot of each formulation of control vater or study chemical in vater is 

kept for analysis. Each stock solution is held in a polyethylene or glass 

carboy and stored at 5°C. For each formulation the quantity prepared vill be 

based on the assumption that each mouse vill consume at most 15 ml per day. 

Standard 500 ml clear, glass, vater bottles are filled vith approximately 

450 ml of control vater or filtered/deionized vater containing one of the 

doses of the test chemical and stoppered. The type of stopper and sipper tube 

may vary vith the toxicity and compatability of the test chemical. The bottle 

is veighed to the nearest 0.1 g and placed into the cage top. After a period 

of seven days, the bottle is removed and reveighed vith the stopper in place. 

The amount consumed in grams per animals is calculated subtracting the second 

veight from the first and dividing the number of animals in the cage. This 

value can then be divided by the number of days the bottle vas in place to 

estimate the vater consumption in grams per animal per day. It is assumed 

that 1.0 g of dosed vater is equivalent to a 1.0 ml volume. A clean vater 

bottle vill then be filled vith the correct dosing solution and stoppered as 

before and placed onto the cage top. Note that the length of time that a 

dosed vater solution can be used is dependent on stability of the test 

chemical. 
P.14 

11 

ANALYSIS OF REPRESENTATIVE DOSING FORMULATIONS 

Samples of dosed feed, drinking vater, or gavage solutions are sent, 

under predetermined shipping conditions, to the reference analysis laboratory. 

Transit time is usually 24 hours and never exceeds 48 hours. Referee aliquots 

are assayed at least once during each task and approximately once a month 

during the continuous breeding phase. Aliquots of the dosing sample sent are 

retained at the RACB laboratory until the analytical report is received. 

ANIMALS 

Animals and Animal Husbandry 

Caesarean-originated Barrier-Sustained (COBS ) Sviss-derived CD-I mice 

are purchased at 6 veeks of age from a barrier facility (Charles River 

Breeding Laboratories, Kingston, NY). A total of 340 mice (170 males and 170 

females) are received on the same date for a given chemical so that Task 1 and 

all subsequent tasks vill be conducted with the same shipment of mice. If 

Task 1 is not to be performed for a particular study, only 120 males and 120 

females are purchased. 

Immediately on arrival, all animals are examined for sickness, 

dehydration, and external body characteristics such as haircoat condition, 

physical abnormalities, sores and/or lesions and evaluated for parasitic 

infection. 

Mice are initially housed 2 per cage in solid bottom polypropylene or 

polycarbonate cages (8" x 11 1/2" x 6") vith stainless steel vire lids (Lab 

Products, Inc., Rochelle Park, NJ). Ab-Sorb-Dri cage litter (Laboratory 

Products, Inc., Garfield, NJ) is used in all cages. Cages are placed on 

galvanized-steel racks (24" V x 60" L x 66" H) vith five adjustable shelves. 

Deionized/filtered vater and pelleted NIH-07 rodent chov (Ziegler Brothers, 
P.15 

12 

Inc., Gardner, PA) are available to both males and females ad libitum. If the 

test chemical is to be delivered via the feed, animals are immediately placed 

on ground rodent chov of the same specifications. Animal holding rooms are 

equipped vith individual temperature controls and automatically adjusted light 

cycles (14:10) (lights on 0700 to 2100 hours). Relative humidity and 

temperature are monitored in the animal holding rooms for each study. 

Temperature is maintained at 21 + 2°C and relative humidity at 30 to 70%. Air 

in each animal room is exchanged a minimum of 12 to 14 times per hour. 

On arrival the animals are placed in quarantine for a minimum of tvo 

veeks to both allow them to adapt to nev surroundings (e.g. noise, 

temperature, humidity, food and caging) and to examine them for diseases. 

During quarantine, tvo males and tvo females from each shipment are sacrificed 

and their sera frozen and sent to Microbiological Associates, Bethesda, MD, 

for determination of antibody titers against 11 mouse viruses. The 11 viruses 

include: Pneumonia Virus of Mice (PVM), Reovirus Type 3, Mouse 

Encephalomyelitis (GDVII), K Virus, Polyoma Virus, Minute Virus of Mice (MVM), 

Ectromelia, Sendai, Mouse Adenovirus, Mouse Hepatitis Virus (MHV) and 

Lymphocytic Choriomeningitis (LCM). Only a batch of animals vhose 

representatives are negative for both endoparasites and the 11 mouse virus 

antibodies are used for RACB studies. 

Each animal receives a unique number (eartag or ear punch) vhich 

identifies both the animal and the particular study. Mice are periodically 

examined for retention of the eartag. If lost, the eartag is replaced by a 

nev tag vith the same number and attached to the opposite ear in order to make 

that animal conspicious to animal observers (National Band and Tag Co., 

Nevport, KY). 
13 

STATISTICAL PROCEDURES USED TO ANALYZE REPRODUCTIVE AND FERTILITY DATA 

Introduction 

Most hypotheses are tested using nonparametric methods. In contrast to 

parametric methods, nonparametric methods require fev assumptions about the 

distribution of the population from vhich samples are dravn. In particular, 

nonparametric methods do not assume that observations come from a normally 

distributed population nor do they require equal variances among treatment 

groups, as do parametric methods. Since nonparametric methods are nearly as 

efficient as parametric methods vhen these assumptions do hold and can be 

substantially more efficient vhen they do not, these procedures provide 

superior control over error rates associated vith hypothesis tests vhen 

distributional assumptions are open to question. Details of the nonparametric 

methods discussed belov can be found in Conover (1971) and Hollander and Uolfe 

(1973). 

Since veight is frequently assumed to be normally distributed, parametric 

methods are used in several analyses concerned vith body and organ veights. 

Description of Statistical Tests 

Most hypotheses are tested using the nonparametric multiple comparisons 

procedures of Dunn (1964) or Shirley (1977), as modified by Williams (1986). 

Shirley's test is designed to detect treatment-related differences vhen the 

response to treatment consistently increases (or decreases) vith increasing 

dose. Although the test employs a smoothing algorithm to adjust for 

dose-response inversions, Dunn's test is more appropriate if the departure 

frora monotonicity is severe. Jonckheere's test (1954) is used to ascertain 

whether there is sufficient evidence of a dose-related response to apply 
P.17 

14 

Shirley's test. If the p-value from Jonckheere's test is less than 0.10, 

Shirley's test is used; othervise, Dunn's test is applied. 

In the crossover mating trial (Task 3), the Kruskal-Wallis test 

(Kruskal-Wallis, 1952) is used to test equality of response among dose groups, 

vhile multiple comparisons tests use the method of Dunn. If Task 4 is 

performed vith only one dose group and the control group, Wilcoxon's test 

(Conover, 1971) is used. 

For data expressed as a proportion, such as number fertile/number 

cohabited, the Cochran-Armitage test (Armitage, 1971) is used to test for a 

dose-related trend, and pairvise comparisons are performed using a chi-square 

test (Conover, 1971). In Task 3, vhere dose groups do not represent 

increasing dose levels, a chi-square test is used to test simultaneous 

equality of response across groups and for pairvise comparisons. 

t 

Since the number of pups in a litter may influence the average pup weight 

in a litter, a parametric analysis of covariance (Neter and Wasserman, 1974) 

is used to test overall equality in average pup veight, after adjustment for 

average litter size. Pairvise comparisons are performed using Dunnett's test. 

Vaginal cytology data are analyzed using a multivariate analysis of 

variance (Morrison, 1976) to test for the simultaneous equality of 

measurements across dose levels. Before applying the test, an arcsine 

transformation is performed to bring the data into closer conformance vith 

normality assumptions. 

Application of Statistical Tests 

Fertility and Mating Indices 

The Cochran-Armitage trend test is used to test for a dose-related 

decrease in fertility and mating indices. Pairvise comparisons betveen 
P.18 

15 

control and dose groups are made using an unadjusted chi-square test. In Task 

3, vhere dose groups do not represent increasing dose levels, a chi-square 

test is used to test for an overall equality among groups and for pairvise 

comparisons. 

Size and Number of Litters 

Litter sizes and number of litters in dose groups are compared to 

controls using Dunn's or Shirley's test. To examine potential differences in 

treatment effects on males and females, number of male pups, number of female 

pups and total number of pups in litters in treated groups are compared to 

controls. 

Proportion of Live Pups and Sex Ratio 

The ratio of the number of pups born alive to the total number of pups 

carried to full term is computed for all fertile pairs. The sex ratio, 

expressed as the proportion of males, is computed for all fertile pairs vith 

at least one live pup. Shirley's or Dunn's test is used to compare dosed 

groups to controls based on Jonckheere's test, as described above. In Task 3, 

Dunn's test is used, and in Task 4 vith tvo groups, Wilcoxon's test is 

applied. 

Pup Body Weight 

To adjust for the potential effect of the number of pups per litter on 

average pup veight, an analysis of covariance is performed. The covariate 

used is average litter size, including live and dead pups. Least squares 

estimates of dose group means adjusted for litter size are computed and tested 

for overall equality using an F-test and pairvise equality using Dunnett's 

test. Unadjusted veights are analyzed vith Shirley's or Dunn's test. To 

examine potential differential effects on males and females, analyses on 

males, females, and both sexes are performed. 
P.19 

16 

Final Task 2 Litter Data 

Average pup veight for the last Task 2 litter of each breeding pair is 

measured at various ages, including birth, veaning and mating. Also, the 

number of pups surviving at each time point, divided by the number of pups 

born alive, is computed for each litter and then the average for each dose 

group is determined. At each time point, dosed groups are compared to the 

controls by Shirley's or Dunn's test. 

Dam Weights 

Dams are veighed immediately following delivery of each litter. 

Shirley's or Dunn's test is applied based on the result of Jonckheere's test, 

as described above. In Task 3, only Dunn's test is used. 

Cumulative Days to Litter 

Task 2 data are analyzed first vith Jonckheere's test and then vith 

either Shirley's or Dunn's test. Task 3 dam veights are analyzed vith Dunn's 

test. Wilcoxon's test is used in Task 4 vhen only tvo groups are to be 

compared. 

Necropsy Weights 

Absolute organ and body veights, and organ veights relative to body 

veight are analyzed by Shirley's or Dunn's test. 

Sperm and Vaginal Cytology Data 

Sperm concentration, sperm motility and percent abnormal sperm are 

analyzed by Shirley's or Dunn's test. An arcsine transformation is applied to 

vaginal cytology data, representing the proportion of the observation period 

that an animal vas in a given estrous stage, to bring the data into closer 

conformance vith normality assumptions. Treatment effects are investigated by 

applying a multivariate analysis or variance to test for the simultaneous 
17 

equality of measurements across dose levels. Cycle length is analyzed using 

Shirley's or Dunn's test. 

Food and Water Consumption 

Using either Shirley's or Dunn's test, food and water consumption data 

are analyzed per animal per day and per gram of body veight per day. 

Cumulative Days to Litter 

The data are first analyzed vith a Jonckheere's test and then vith either 

a Shirley's or Dunn's test depending on the results of the Jonckheere's test. 

Treatment effects are investigated by applying a multivariate analysis of 

variance to test for the simultaneous equality of measurements across dose 

levels. Cycle length is analyzed using Williams's test. 

METHODS - SPECIFIC 

TASK 1 - DOSE RANGE FINDING STUDY (See Figure 2 and the milestone chart for a 

composite time line) 

Selection of Animals for Task 1 

A total of 100 animals are used in Task 1; 96 for treatment and four for 

quality control. One veek after arrival, each of the 340 animals purchased 

(170 males and 170 females) is eartagged and assigned a random number from a 

table of random numbers (Snedecor and Cochran, 1967). The 50 males vith the 

highest random numbers vill be assigned to Task 1. Of these 50, the tvo vith 

the highest random numbers vill be used for quality control. The same process 

vill be folloved for the assignment of Task 1 females. 

Randomization into Treatment Groups in Task 1 

The animals used in Task 1 are veighed at the conclusion of veeks 1 and 2 

of quarantine. If no health problems are observed during quarantine, Task 1 
P.21 

18 

animals vill be ranked by the veek 2 body veights and assigned to treatment 

groups by a stratified randomization process so that the mean body veights of 

each group are statistically similar. 

Pairing of Animals 

Tvo animals of the same sex are housed per cage during Task 1. To select 

cagemates vithin a treatment group, previously assigned random numbers are 

arranged in ascending order, males and females separately. The male/female 

vith the lovest random number is paired vith the male/female vith the next 

higher random number. All animals are paired in this manner. 

Study Results 

The folloving data are collected and evaluated during Task 1: 

1. Body veight on days 0, 7, and 14. 

2. Average daily intake of feed and drinking vater for each veek in 

order to assess test agent intake. 

3. Mortality during the 14 days of treatment. 

4. Clinical symptoms of toxicity (tvice daily). 

The percent veight change over the tvo-veek period is analyzed by a 

tvo-vay analysis of variance (dose vs. sex) and percent mortality is analyzed 

by the Chi-Square Test. LD is determined based on the mortality data. The 

probit analysis is done to determine LD1 Q, if death occurs in tvo or more 

groups (Snedecor and Cochran, 1967). In addition to the probit analysis, the 

change in body veight is considered. From these data, the MTD (maximum 

tolerated dose), defined as the highest dose that can be administered for tvo 

veeks vithout depressing veight by more than 10% or resulting in significant 

mortality (>10%), is computed. These data are then utilized to calculate the 

doses for Task 2 so that the highest dose vill not depress weight by more than 

10% over the 98-day continuous breeding study. 
P.22 

19 

It should be noted that if sufficient information is available in the 

literature or from previous studies to estimate the MTD, Task 1 is not 

conducted. 

TASK 2 - CONTINUOUS BREEDING 

Quarantine 

The animals for Task 1 and 2 are usually purchased at the same time; 

therefore, the animals are held in isolation for five veeks prior to 

commencing Task 2, (i.e. tvo veeks preceding Task 1, the tvo-veek period 

during vhich Task 1 is conducted and one veek during vhich the Task 1 data are 

being analyzed, and the dose levels for Task 2 selected). Mice may also be 

purchased separately for Task 2 in vhich case they receive the usual 2 veek 

quarantine period. 

Randomization Into Treatment Groups 

The 240 animals, 120 males and 120 females, maintained in quarantine 

through the conclusion of Task 1 are reveighed and assigned to treatment 

groups by a stratified randomization procedure based on veight as exemplified 

by the folloving example. The animals of each sex are ranked according 

to veight. Surplus animals (only 100 males and 100 females vill be utilized) 

are removed from the top and bottom of the random number list. Beginning at 

the top of the list of randomized animals the males are divided into subgroups 

of five each. Of these five males, the tvo animals vith the highest random 

numbers are assigned to the control group; the animal vith the third highest 

goes into the lov dose group, the fourth highest to the mid dose and the 

lovest to the high dose group. This process is repeated vith the next five 

animals and so on until all males have been placed into a treatment group (40 
P.23 

20 

in control and 20 in each of the groups receiving the test chemical). The 

same procedure is used to assign females to the four treatment groups (one 

control group vith 40 females and three dose levels vith 20 females per dose). 

Initiation of Treatment vith Test Chemical: Week 1 

Animals are 11 veeks of age at the initiation of treatment. During the 

first seven days of treatment, the sexes are housed separately in groups of 

tvo in 7 1/2" x 11 1/2" x 5" polypropylene or polycarbonate cages. The test 

chemical is administered via gavage, feed or drinking vater. 

Continuous Breeding (Cohabitation) for 98 Days 

Folloving treatment vith the test chemical for seven days, one male and 

one female are selected randomly (see belov) from the same dose group and 

cohabited for 98 days (14 veeks). There are 40 breeding pairs in the control 

group and 20 pairs in each group receiving the test chemical (three dose 

levels) for a total of 200 mice (100 males and 100 females). The animals vill 

be 12 veeks of age at the commencement of cohabitation. 

Random Selection of Breeding Pairs 

To select breeding pairs, the male vith the lovest random number vithin a 

treatment group is paired vith the female vith the lovest random number. The 

male vith the second lovest random number vith the corresponding animal from 

the female list, and so on. 

Housing and Identification of Breeding Pairs 

Each breeding pair is assigned a number vhich is placed on the cage card 

displaying the number of the male and female and a color coded dose level 

indicator. The pairs vill be housed in 7 1/2" x 11 1/2" x 5" polycarbonate 

cages. Othervise, housing conditions are the same as those described 

previously. 
P.24 

21 

Dose Selection for Task 2 

The data from Task 1 are utilized to determine the appropriate doses for 

Task 2. Doses are selected as follovs: The high dose is selected so that it 

vill produce some general toxicity (i.e. maximal tolerated dose) as defined by 

specific organ toxicity (depending on the chemical) or body veight depression 

of about 10% over the course of the study. The mid-dose is expected to 

produce no systemic toxicity and the lov dose is an estimated no-effect level. 

Data Collection During the 98-Day Cohabitation Period 

The folloving criteria are evaluated during Task 2: 

1. Body Weight - All experimental animals are veighed on day - 0, 7, 14, 

21, 35, 63, 91 and 126 (beginning of veeks 1, 2, 3, 5, 9, 13, and 18, 

respectively). Refer to milestone chart in appendix for generic 

veigh dates. 

2. Pups Delivered During Days 0 to 97 - All cages are inspected at least 

tvice a day to check for the presence of pups. After the dam has 

completed delivery, the pups are counted, sexed, and veighed as 

described later in this section. The pups are then sacrificed by 

asphyxiation vith carbon dioxide. If a female has not completed 

delivery by evening health surveillance, the pups are checked the 

next day and this day vill be considered postnatal day 0. In the 

event a pregnant female mouse dies prior to or during parturition, an 

immediate necropsy is performed and, if possible, the number of live 

or dead pups in the uterus is counted. 

3. Evaluation of Pups - The day of delivery is designated as postnatal 

day 0. After the dam has delivered and has cleaned her pups, she is 

veighed. The veight and time is recorded. The total number of pups, 

number of live pups, and minimum number of dead/cannibalized pups is 
P.25 

22 

noted. All pups are examined for external abnormalities. Live pups 

are sexed. The sex of a pup is determined externally by the distance 

betveen the urogenital and anal openings (male distance is tvice the 

female distance, usually 2 mm vs. 1 mm) and male perineum appears 

slighly svollen (site of future scrotal sacs). Within a litter, all 

males are placed on the balance and the veight is recorded in grams 

to tvo decimal places. The same procedure is folloved for female 

pups. Dead/cannibalized pups are not sexed or veighed. A tissue is 

placed on the balance to prevent chilling of pups. 

4. The number of days to litter is calculated for each breeding pair. 

5. Dam veights are measured after each litter. If the route of test 

chemical is by gavage, the dam veight is used for determining dosage 

volume until the next veighing session is scheduled. 

6. An aliquot of each of the representative dosing solutions is 

submitted for reference chemical analysis. The specific veeks vhen 

samples are submitted are 1, 6, 10, and 14. 

7. Dosed feed/vater consumed is monitored for a veek once a month 

throughout cohabitation (veeks 1, 2, 5, 9, 13, and optionally 18). 

Separation of Breeding Pairs 

Immediately folloving the 98-day cohabitation period, each pair is 

separated and the male and female are housed separately in cages of the same 

dimensions and othervise identical conditions as the 98-day cohabitation 

period. All animals are continued on their respective treatments for a period 

of 21 days. 

Pups delivered during the holding period are alloved to remain vith their 

dams. These offspring are counted, sexed, and veighed on days 0, 4, 7, 14, 

and 21 and the percent survival is calculated. Post partum dam veight is also 

recorded. 
23 

Disposition of Animals 

If no effect on reproductive performance is observed, then 

a) Randomly selected offspring of the control and high-dose animals are 

utilized for Task 4. The other litters are humanely sacrificed vhen 

they are 21 days of age. 

b) The control lov, mid and high-dose animals are humanely sacrificed. 

If an effect of reproductive performance is observed, then: 

a) Task 3 is conducted vith the control and high dose animals after 

vhich they are necropsied, 

b) The lov and mid-dose animals are humanely sacrificed. 

c) Randomly selected offspring of the control, lov, mid as veil as high 

dose animals are utilized for Task 4. 

Statistical Evaluation of Task 2 

Data vill be totaled and summarized veekly and at the conclusion of the 

98-day cohabitation period. 

The breeding pairs are the independent sampling unit and reproduction 

parameters for each pair are summed across litters in order to carry out 

statistical analysis of the data (See Statistical Procedures, p. 13). 

TASK 3 (DETERMINATION OF THE AFFECTED SEX) 

When the last litter of Task 2 is 21 days old, the pups are veaned. If 

an effect on reproduction is observed in Task 2, Task 3 is then conducted 

using the controls and high-dose animals. Mid-dose animals may be used if the 

effect of the toxicant vas severe in the high-dose group (i.e. no litters at 

all or only a first or second litter or high mortality or severe generalized 

toxicity) and the mid-dose animals shoved a significant reduction in 

fertility. 
24 

Random Assignment of Breeding Pairs 

Of the 40 control males and 40 control females, 20 of each sex are used 

for a control x control cross and 20 of each sex are bred with high dose 

animals. The 20 control females vith the highest random numbers (either 

assigned earlier for Task 2 or a nev random number) and 20 control males vith 

the lovest random numbers are used for the control x control mating. The 20 

lovest females and 20 highest males are used for breeding vith the high dose 

animals. The breeding pairs are determined by a randomization procedure 

similar to that of Task 2 (p. 18). 

Breeding for Task 3 

Breeding pairs are checked tvice daily for the presence of a copulatory 

plug. Cohabitation of breeding pairs continues for seven days or detection of 

a copulatory plug, vhichever is first. Animals are not exposed to the test 

chemical for this period of cohabitation. Exposure is reinstated after seven 

days and continues until the conclusion of the Task. Folloving separation the 

male and female are housed separately (as for Task 2 last litter) until at 

least day 28 of Task 3. During this period fertility data are collected on 

any litters produced as described for Task 2 and the litter are sacrificed. 

Postpartum dam veights are determined after delivery of the litter. In 

addition, all animals are veighed on the first day of cohabitation and once a 

veek thereafter until sacrifice and the amount of feed/vater consumed is 

monitored veekly. The folloving information is also calculated: 

Fertility index = (No. fertile/No. cohabited) x 100 

(No. fertile if defined as no. producing at least one litter) 

Mating index = (No. vith copulatory plugs/No. cohabited) x 100 

Randomization of Animals Prior to Scheduled Necropsy at the End of Task 3 

A random number is assigned to each animal scheduled for necropsy. 

Random numbers are sorted in ascending order. This list represents the order 

of necropsy. 
P.28 

25 

Task 3 (Necropsy) 

After Task 3 litters have been sacrificed the male and female mice are 

necropsied in the presence of a pathologist. The pathologist examines the 

entire animal for abnormalities. The complete procedure should take no longer 

than tventy minutes for each animal. 

During the scheduled male necropsy, the folloving parameters are 

evaluated: 

1. Sperm motility, sperm density (cauda epididymal sperm number/mg 

tissue), and sperm morphology. 

2. Body veight. 

3. Liver veight. 

4. Right testis veight. 

5. Ventral prostate veight (pair). 

6. Seminal vesicle veight (pair including the coagulating glands). 

7. Epididymal veight. 

8. Combined kidney (left and right) veight vith adrenal gland attached. 

9. Other selected organ veights, based on the specificity of the test 

chemical, may also be measured. 

After the animal is veighed, the prostate, seminal vesicles epididymis 

and testis vith attached epididymis is excised, veighed and fixed in Bouins 

for embedding in paraffin or in 10% neutral buffered formalin if they are to 

be embedded in glycol methacrylate. The liver and kidneys are fixed in 10% 

neutral buffered formalin. 

For the females, estrous cyclicity is measured by vaginal smears for 12 

days prior to necropsy. The folloving parameters are then evaluated during 

necropsy: 
P.29 

26 

1. Body veight. 

2. Liver veight. 

3. Combined kidney (left and right) veight vith adrenal glands attached. 

4. Right ovary. 

5. The reproductive tract is excised and fixed in 10% neutral buffered 

formalin but not veighed. The right ovary from each female is fixed 

in Bouin's solution. Histopathology of selected tissue is conducted 

as/when needed. 

Sperm Motility Determination 

Sperm motility is extremely sensitive to pH and temperature. Therefore, 

the folloving materials are assembled and maintained at 37°C: 

phosphate buffer 

microscope slides and coverslips 

Pasteur pipets 

petri plates 

microscope stage varmer 

modified Tyrode's egg yolk solution adjusted to 7.2. pH (Environmental 

Health and Testing, Lexington, KY). 

After the right cauda epididymis is excised and veighed. The tissue is 

placed in a dish vith saline at 37°C and picked vith a forceps to release some 

sperm. Sperm motility is assessed as quickly as possible. A small drop of 

seminal fluid is pressed into a drop (80ul) of Tyrodes solution on a prevarmed 

microscope slide. Tvo independent slides are prepared to estimate sperm 

motility. 

A varm coverslip (24 x 40 mm) is placed on the slide and the slide is 

placed on a varmed microscope stage (Bailey Instruments, Inc., Saddle Brook, 

NJ) to maintain the sperm at 37° during the motility evaluation. 
P.30 

27 

To estimate sperm motility, the actual number of motile and nonmotile 

sperm for each field of vision is determined. A hand counter is used to count 

the number of motile and nonmotile sperm. The final motility is based on ten 

independent fields and observations made by tvo independent vievers using a 

40X objective. A minimum of 200 total sperm must be observed in males vith 

lov sperm counts. 

Sperm are considered motile if they shov any movement at all. Since 

percent motility decreases vith time, evaluation time is critical and must be 

accomplished as quickly as possible (i.e., 3-5 min.). Similarly temperature 

and pH must be carefully controlled. 

Sperm Count 

After the slides for sperm motility evaluation have been prepared, the 

entire cauda is immediately placed into 2.0 ml of phosphate buffered saline 

(PBS) (pH 7.4) in a 35 x 10 mm plastic petri dish. The cauda is gently minced 

vith a scalpel, thereby releasing the caudal spermatozoa. The petri dish 

contents are svirled several times and placed in an incubator (37°C) for 15 

minutes in order to allov time for aggregates of sperm to disperse. 

At the conclusion of incubation, the contents are svirled again to evenly 

distribute the sperm suspension and 0.5 ml of the sperm suspension is 

transferred to a test tube and diluted vith PBS. The volume of Tyrode's 

solution added may be varied according to the density of the sperm suspension. 

The sperm are then killed by placing the test tube in boiling vater for one 

minute and one drop of the suspension is placed in each of tvo hemocytometer 

chambers. 

Tvo counts are performed using separate samples of diluted sperm 

suspension. Consistency in the preparation of sperm samples for density 

determinations is essential to obtain accurate results. 
P.31 

28 

The total number of sperm per cauda epididymis and per mg of cauda 

epididymis is then calculated. 

Sperm Morphology 

The procedures and criteria of Wyrobek and Bruce (1975) and Soares et al. 

(1979) are used to evaluate sperm morphology. The suspension of sperm is 

transferred to a test tube and one drop of 1% Eosin Y stain in water is added. 

The contents of the tube are gently agitated and allowed to stain for 

approximately 45 minutes. The sperm are gently resuspended with a Pasteur 

pipet, placed on a microscope slide. Four slides are prepared from each sperm 

suspension. After smears have been made, the slides are placed at a 30 to 40 

degree angle on a tray so that excess suspension can drain off. The slides 

are air-dried overnight in a dust-free atmosphere and coverslipped using 

Permount or suitable mounting medium and xylene. Five hundred sperm are 

evaluated per sperm suspension (125 sperm per slide is a minimum requirement) 

at 400X magnification. There are seven categories for sperm morphological 

evaluation: normal; blunt hook; banana; amorphous; pin-head; tvo heads/tvo 

tails; and tailless sperm. The data are reported as percent abnormal sperm 

per sample. 

Vaginal Cytology Evaluation 

Vaginal smears obtained from mice have been found to correlate veil vith 

changes occurring in the reproductive tract and vith the secretion of the 

ovarian hormones. The most common types of cells present in a vaginal smear 

are: 

a. Polymorphonuclear leucocytes 

b. Nucleated epithelial cells 

c. Large squamous epithelial cells, also called cornified cells 

(nucleated or non-nucleated) 
29 

The estrous cycle of the mouse is completed in four to five days although 

the timing of the cycle may be influenced by exteroceptive factors (light, 

temperature, etc.). The cycle is roughly divisible into four main phases. 

Estrus - This is the period of "heat", and copulation is possible only 

at this time. This phase lasts from 9 to 15 hours and is characterized 

by a high rate of running activity. Many mitoses occur in the vaginal 

mucosa, and as nev cells accumulate, the superficial layers become 

squamous and cornified. The latter cells are exfoliated into the vaginal 

lumen and their presence in vaginal smears is indicative of estrus. 

During late estrus there are "cheesy masses" of cornified cells vith 

degenerate nuclei present in the vaginal lumen, but fev, if any, 

leucocytes are found during estrus. Much of the luminal fluid in the 

uterus is lost before ovulation. 

Metestrous - This occurs shortly after ovulation and is intermediate 

betveen estrus and diestrus. This phase lasts for 10 to 24 hours and 

mating is usually not permitted. Many leucocytes appear in the vaginal 

lumen along vith a fev cornified cells. 

Diestrus - This phase lasts for 60 to 70 hours, during vhich functional 

regression of the corpora lutea occurs. The vaginal mucosa is thin and 

leucocytes migrate through it, giving a vaginal smear consisting almost 

entirely of leucocytes. 

Proestrus - This heralds the next "heat" and is characterized by 

functional involution of the corpora lutea and preovulatory svelling of 

the follicles. Fluid collects in the uteri and they become highly 

contractile. The vaginal smear is dominated by nucleated epithelial 

cells vhich occur singly or in sheets. The duration is approximately 12 

hours. 
P.33 

30 

1. Obtaining a Vaginal Smear from Mice 

A vaginal smear from each animal is prepared betveen 8 and 10 a.m. 

for 12 consecutive days before terminal sacrifice. Fully frosted slides 

are used to make vaginal smears (Dakin-Erie, Cat. #2958). All smears are 

made on the frosted front of the slide. Six days of smears are 

incorporated on one slide vhich can be stained at one time. One drop of 

0.9% saline or PBS solution is placed on the microscope slide in the 

appropriate square. Next, a small amount (-50 pl) of 0.9% saline or PBS 

solution is aspirated into a 3-4" medicine dropper. The medicine dropper 

is placed just inside the vaginal opening of the mouse and the vaginal 

fluids lavaged several times. 

The contents of the medicine dropper are transferred onto the slide and 

alloved to air-dry. If a non-disposable eye dropper is used, it is thoroughly 

vashed betveen animals in 95% alcohol folloved by saline. At the end of each 

day, the smears are fixed by lightly spraying fixative (Spray Cyte II by Clay 

Adams, #7180). In order to fix only the specific square on the slide, a 2" x 

4" file card vith a vindov is used. After all smears have been made, the 

slides are alloved to dry completely and stained vith Toluidine Blue the same 

day or vithin the next fev days. 

For staining, slides are loaded in glass racks and dipped in the 

folloving solutions: 

- 95% Ethanol - 30 minutes 

- 80% Ethanol - 1 minute vith gentle shaking 

- 70% Ethanol - 1 minute vith gentle shaking 

- 50% Ethanol - 1 minute vith gentle shaking 

- 20% Ethanol - 1 minute vith gentle shaking 
P.34 

31 

- 0.5% Toluidine Blue in 20% Ethanol - 30-45 seconds 

Personal judgement should be used because Toluidine Blue improves with 

age. 

- Running tap water rinse - until tap water runs clear (at this point, 

if slides are not dark enough, they are dipped in 20% Ethanol and put 

back in the stain. This process is repeated until desired color is 

obtained). Adequacy of staining should be judged based on the 

metestrous or diestrus smears. 

The stained slides are gently blotted vith bibulous paper. Papers are 

not reused because material on a slide can be tranferred to other slides. 

Slides are alloved to dry completely and then coverslipped using Permount (or 

another suitable mounting medium) and xylene. 

Evaluating Vaginal Smears 

The slides are coded and evaluated using a lov pover objective for 

scanning and a 20X objective to make the final judgement. Each smear is 

assigned to one of the estrous stages (proestrus, estrus, metestrus, diestrus) 

as previously defined. When it is difficult to make any judgement from the 

smear, it is designated 'not clear.' 

The estrous cycle length is estimated as follovs: the number of days 

betveen tvo consecutive proestrus (P) stages beginning vith the first P are 

counted. The second P is not counted as a day in the cycle length; it is the 

beginning of the next cycle. 

- When tvo Ps are not present, tvo nonconsecutive Estrus Stages are used 

folloved by tvo Metestrus Stages, then tvo Diestrus Stages. 

- The estrous cycle length is considered not clear vhen an animal is in 

the same estrous phase for seven consecutive days. 
P.35 

32 

Pathologic Observations 

At necropsy, in addition to the detailed examination of the reproductive 

organs, gross lesions of all tissues are noted. A complete histopathological 

examination of the liver, kidney, reproductive tissues and other selected 

tissues may be performed. 

TASK 4 (OFFSPRING ASSESSMENT) 

If chemical exposure caused no significant effects on fertility and 

reproductive performance in Task 2, then randomly selected members of each sex 

from the final litter (i.e. the litter born during the holding period after 

the 98-day cohabitation period in Task 2) from the control and high dose group 

are raised until 74 + 10 days of age. 

If the chemical vas shovn to be a reproductive toxicant, then randomly 

selected members of the final litter from the control and all treatment groups 

are saved and raised until 

74 + 10 days of age. 

Animals are dosed vith the same test article as their parents from 

veaning throughout Task 4. 

Random Selection of Weanlings for Offspring Assessment 

All animals in the last litters of each treatment group are not needed 

for the Task 4 assessment; therefore, at 21 days of age all pups are assigned 

a random number. In general, vithin each litter, random numbers are then 

arranged in descending order, males and females separately. The tvo male pups 

vith the highest random numbers are selected to conduct Task 4. The tvo 

female pups vith the highest random numbers are selected similarly. 
P.36 

33 

Pup mortality is closely monitored prior to and after veaning. If the 

pup mortality in the treated group appears to be significantly higher than the 

control group, more than tvo pups of each sex may be saved from each litter. 

Also, more animals may be selected from a litter if the chemical is 

administered via gavage, rather than the feed or vater. Frequently, dosing 

errors occur by gavage, resulting in the death of the weanling. The animals 

not selected from the litters are killed by asphyxiation with C02 . 

Housing Randomly Selected Weanlings 

Weanlings are housed tvo or three per cage by sex and treatment in 

7 1/2" x 11 1/2" x 5" polycarbonate cages under the conditions described 

previously in Task 1. When only one pup per sex per litter is selected, 

non-littermates of the same sex and treatment are housed together. If tvo or 

four animals per sex per litter are selected they are housed in pairs by sex 

and treatment as littermates. If three animals per sex per litter are 

randomly selected, tvo of them are housed together. The third veanling will 

be housed singly, if no other litters from that treatment group are to be 

veaned that day, or paired vith an animal from a different litter of the same 

sex and treatment. All Task 4 animals are held in this manner, and continue 

to receive treatment until 74 + 10 days of age. 

Task 4. Random Selection of Ft Mating Pairs 

Random numbers previously assigned to select pups for the Task 4 study 

are also used to select mating pairs vhen the animals are 74 + 10 days old. 

The male and female pups vith the tvo highest random numbers are chosen to 

represent each litter. It is preferred that tventy (20) male and tventy (20) 

female pups vithin a group represent tventy (20) different litters. Since 

this may not alvays be possible, a second male (or female) pup may be selected 

from a litter. One vay to form pairs is as follovs. Animals are paired 
P.37 

34 

vithin a treatment group by matching the male vith the highest random number 

to the female vith the highest random number and so on. Sibling matings are 

avoided. For example, if the male with the third highest random number is 

sibling to the female with the third highest random number, it is paired with 

the female vith the fourth highest random number. Similarly, the female mouse 

vith the third highest number is mated vith the male vith the fourth highest 

random number. 

Task 4. Breeding Assessment 

All breeding pairs are housed as described for Task 3. They are 

separated after seven days or upon observation of a vaginal copulatory plug, 

vhichever is first, and housed separately for 21 days folloving conclusion of 

the cohabitation period. During this period, fertility data are collected on 

any litters produced as described for Task 2 and the litter is sacrificed. 

Postpartum dam veights are measured after delivery of the litter. In 

addition, all animals are veighed on the first day of cohabitation and once a 

veek thereafter until necropsy and the amount of dosed feed/drinking vater 

consumed is monitored on a veekly basis until necropsy. Fertility index and 

mating index is also calculated. 

Randomization and Necropsy 

The ?1 parents are randomized for necropsy and necropsied as detailed for 

Task 3. To assess estrous cyclicity, a vaginal smear is obtained daily for 

the last 12 days prior to sacrifice of the females. 

Statistical Analysis 

(See pages 12-16.) 
P.38 

35 

8. RESULTS 

Summary Data from RACB Studies 

The folloving section details in overviev fashion all the results 

obtained from a RACB study. In order to better understand hov this data is 

organized and utilized in an actual study, it may be helpful to examine an 

NTIS technical report from a RACB study. 

1. Analysis of dosing samples (Quality Control) 

Task 1 - Week 1 

Task 2 - Week 1 

Task 2 - Week 4 

Task 2 - Week 8 

Task 2 - Week 12 

Task 2 - Week 16 

Task 3 - Week 23 

Task 4 - Week 20 

Task 4 - Week 24 

2. Summary of body veight and daily feed/vater consumption for Task 1 by 

dose and sex 

Percent mortality 

Percent change in body veight 

Estimated daily dose level of the test article 

Body veight on days 0, 7 and 14 

Number of live animals on days 0, 7 and 14 

Average daily consumption of dosed feed/vater for veek 1 and 

veek 2 

3. Fertility of pairs during Continuous Breeding (Task 2) 

Treatment group vs. Fertility index 
P.39 

36 

4. Reproductive performance of fertile pairs during Continuous Breeding 

(Task 2) by treatment group 

Litters/pair 

Live pups/litter 

Proportion of pups born alive 

Sex of pups bom alive 

Live pup veight - male, female combined 

Live pup veight adjusted for litter size 

5. Postpartum dam veight (Task 2) 

6. Cumulative days to litter Task 2/pair 

7. Mating and fertility of pairs after a mating trial to determine 

affected sex 

Control x Control 

Mating Index and Fertility Index Control M x Treated F 

Control F x Treated M 

8. Reproductive performance of fertile pairs after a mating trial to 

determine affected sex. 

Litters/pair 

Live pups/litter 

Proportion of pups born alive 

Sex of pups born alive 

Live pup veight - male, female, combined 

Live pup veight adjusted for litter size 
P.40 

37 

9. Female body and organ veights at necropsy (Task 3) Actual and adjusted 

for body veight 

Body 

Liver 

Kidney 

10. Male body and organ veights at necropsy (Task 3) Actual and adjusted for 

body veight 

Body (g) 

Liver (mg) 

Kidney (mg) 

Seminal vesicle (mg) 

Testis (mg) 

Epididymis (mg) 

Cauda epidydimis (mg) 

Prostate (mg) 

11. Summary of data from sperm studies (Task 3) 

Sperm motility (%) 

Sperm concentration/mg caudal epididymis 

Abnormal sperm (%) 

12. Summary of data from vaginal cytology studies (Task 3) 

Relative frequency of estrous cycles/stages/group 

13. Mating and fertility of second generation breeding pairs (Task 4) 

Mating Index and Fertility Index 

14. Reproductive Performance of Second Generation Breeding Pairs (Task 4) 

Litters/pair 

Live pups/litter 

Proportion of pups born alive 

Sex of pups born alive 

Live pup veight - male, female combined 

Live pup veight adjusted for litter size 
P.41 

38 

15. Second generation female body and organ veights at necropsy (Task 4) 

Actual and adjusted for body veight 

Body (g) 

Liver (mg) 

Kidney (mg) 

Right ovary 

16. Second generation male body and organ veights at necropsy (Task 4) 

Actual and adjusted for body veight 

Body (g) 

Liver (mg) 

Kidney (mg) 

Seminal vesicle (mg) 

Testis (mg) 

Epididymis (mg) 

Cauda epidydimis (mg) 

Prostate (mg) 

17. Summary of data on sperm studies (Task 4) 

Sperm motility (%) 

Sperm concentration/mg caudal epididymis 

Abnormal sperm (%) 

18. Summary of data on vaginal cytology studies (Task 4) 

Relative frequency of estrous cycles/stages/group 
P.42 

39 

19. Body veight data (pair) (FQ animals) 

Week 0 

Week 1 

Week 2 

Week 3 

Week 5 

Week 9 

Week 13 

Week 18 

Week 22 

Week 23 

Week 24 

Week 25 

20. Daily feed/vater consumption/pair (FQ animals) 

Week 1 

Week 2 

Week 5 

Week 9 

Week 13 

Week 18 

Week 22 

Week 23 

Week 24 

Week 25 
P.43 

40 

21. Second generation body veight and survival data (Final Task 2 litter) 

Body veight on days 1, 4, 7, 14, 21 of age and 10, 11, 12, and 13 

veeks of age 

22. Second generation daily feed/vater consumption 

Weeks 10, 11, 12, and 13 of age 

23. The pathology report provides a summary of the results of the gross and 

histopathologic findings and any conclusions about the effects of the 

test chemical on the animals' organs. The folloving tables are included 

vith this section: 

1. Correlation of gross and histopatologic changes for each animal 

affected. 

2. The incidence of histopathologic change in the organs affected by 

treatment group. 

Individual Animal Data 

1. Individual initial and final body veights and litter data for breeding 

pairs (Task 2) 

2. Reproductive performance of individual pairs after a mating trial 

(Task 3) 

3. Individual animal necropsy data (male and female) (Task 3 and 4) 

Body veights 

Organ veights 

Sperm parameters 

Vaginal cytology (estrous cycle length) 

4. Reproductive performance of individual pairs of second generation mice 

(Task 4) 
P.44 

41 

CONCLUSION 

The RACB protocol provides a comprehensive approach to the question of 

reproductive toxicity. Results from studies of compounds tested at tvo 

laboratories indicate that the RACB protocol effectively identifies toxicants 

affecting fertility, mating, prenatal viability, fetal grovth in utero, gender 

specific prenatal toxicity, toxicity for both the FQ and Fx generation, and 

toxicity during the preveaning and postlactational periods for Fx offspring, 

in addition to generalized toxicity and treatment-specific toxicity to organs 

of interest in both sexes of FQ and Fx generation. In addition, the RACB 

protocol provides the opportunity to observe progressive changes in fertility 

(vith successive litters) after continuous exposure of monogamous breeding 

pairs to the test compound. In short, through the efforts of the National 

Toxicology Program and their contracting laboratories, a valuable reproductive 

toxicity study design has been developed, refined, and proven sensitive, 

effective and useful. The basic protocol delineated in this volume has been 

utilized virtually unmodified for the testing of over 50 chemicals. Hovever, 

these procedures should not be vieved as absolute. As the results of the 

initial studies are assessed, it is anticipated that pitfalls, problems and 

alternative or supplemental tests vill become apparent vhich vill result in 

modifications both minor and substantial to this protocol. 

The interested reader is urged to contact the National Toxicology 

Program's Developmental and Reproductive Toxicology Group for the latest RACB 

protocol. Several RACB studies have been published and these can be consulted 

in order to see hov the data is actually organized into a publication (Chapin 

et al., 1988, Lamb et al., 1985, Lamb et al., 1987, Heindel et al. 1989, and 

Morrissey et al., 1988). In addition, copies of any of the final reports can 

be obtained through NTIS (see references) for a nominal fee. 
P.45 

Reproductive Assessment By Continuous Breeding 

Row Diagram - Current Design 

TASK 4 

OlfapHng Aaaaoamartt 

Pt QaoaTBtlon) 

(Control * 1 doaoa: group) 

TASKS 

OotonoiiutloM ol 

AIIOBIOO 9mm 

(F, Oofwrotlo* 

TASK 4 

Offspring AHOMfflont 

P< Oanorallon 

(Contral 4 1 doaod groups) 

NECROPSY 

Sporm Moiphology 

Vaginal Cytology 

optional 

NECROPSY 

Sporm Morphology 

Vaginal Cytology 

optional 

NECROPSY 

Sporm Morphology 

Vaginal Cytology 

optional 

HtatopatHotegy 

Hormono Aaaaya 

Htatopathology 

Hormono Aaaaya 

Hlatopathology 

Hormono Aaaaya 

FIGURE 1 
REPRODUCTIVE ASSESSMENT BY CONTINUOUS BREEDING 

POSITIVE STUDY: 

Control F-H | 

-*» B E 

Middle B E 

High Qb^ . 

v 

• v 

ft 

M 

V 

I v» 

MC 

^ r 

C C I | 1 TASK 3 

C » H | | 1 * * Creaaovor 

. ** 

TASK 1 

Dose 

RangeFinding 

TASK 2 

Continuous Breeding 

Holding 

Period; 

TASK 4 

Socond Generation 

Rearing 

0 2 3 

i I r— 

17 20 23 

i i i 

31 34 

NEGATIVE STUDY 

Wooka 

Control 

^ S ^ ^ - . ** 

Low F=3P" 

•^—*—*—*— r 

Middle R F 

•^ * ^ S * 

High £3 E 

•^—r 

M .** 

TASKJ 

Ooa« 

RangeFinding 

TASK2 

Continuous Breeding 

Holding TASK 4 

Period! Second Generation 

Rearing 

i i i 

0 2 3 17 

- r ~ 

20 

-r— 

23 

Week* 

34 

Fig. 2. Time line schematic of the RACB protocol for positive and negative 

studies. * indicates sacrifice. ** indicates complete necropsy. 

'—-• indicates males and females housed separately and I— ' indicates males 

and females housed as breeding pairs. Arrows indicate litters born and M 

signifies the mating period for Task 3 and 4. 
MILESTONE CHART 

Major Tasks in the RACB Protocol and the Associated Sequence of Events 

for a Generic Compound vith a Theoretical Start Date of January 1 

42 

MILESTONES EVENT DATE 

QUARANTINE: 

Animals Arrive 

Ear Tag Animals (end of Veek 1) 

Weigh Animals, Randomize, and Assign Random Numbers 

for Task 1 (1 to 2 days before start of Task 1) 

TASK 1: 

January 1 

January 8 

January 13 

Start Dosing, Weigh Animals, Weigh Nev Food and Water 

(day 0 of Task 1) 

Weigh Animals, Weigh Nev and Old Food and Water 

(end Week 1) 

Weigh Animals, Weight Old Food and Water (end Week 2) 

Analyze Task 1 Data and Select Task 2 Dose Groups 

(after Task 1 is complete) 

TASK 2: 

Weigh Animals, Randomize for Task 2 (1 to 2 days 

before start of Task 2) 

Start Task 2 - Weigh Animals, Weigh Nev Food and Water 

(day 0 of Task 2) 

Pair Animals, Weigh Animals, Weigh Nev and Old Food 

and Water (end Week 1) 

Weigh Animals, Weigh Old Food and Water (end Week 2) 

Weigh Animals, Nev Food and Water (start Week 5) 

Weigh Animals, Weigh Old Food and Water (end Week 5) 

Weigh Animals, Nev Food and Water (start Week 9) 

Weigh Animals, Weigh Old Food and Vater (end Week 9) 

Weigh Animals, Nev Food and Water (start Week 13) 

Weigh Animals, Weigh Old Food and Water (end Week 13) 

Separate Pairs (end Veek 15) 

Weigh Animals, Nev Food and Water (start Week 18) 

Weigh Animals, Weigh Old Food and Water (end Week 18) 

Wean F1 Animals (veeks 3 to 6 after separation of pairs) 

January 15 

January 22 

January 29 

January 30-

February 2 

February 3 

February 5 

February 12 

February 19 

March 5 

March 12 

April 2 . 

April 9 

April 30 

May 7 

May 21 

June 4 

June 11 

June 11-

July 1 
44 

REFERENCES 

Armitage, P. (1971) Statistical Methods ni Medical Research. John Viley & 

Sons, Nev York. 

Chapin, R.E., George, J.D., and Lamb, J.C, IV. (1988) Reproductive Toxicity 

of Tricresyl Phosphate in a Continuous Breeding Protol in Sviss (CD-I) 

Mice. Fund, and Appl. Toxicol. 10, 344-354. 

Conover, V.J. (1971) Practical Nonparametric Statistics. John Viley & Sons, 

Nev York. 

Dunn, O.J. (1964) Multiple Comparison Using Rank Sums. Technometries 6, 

241-252. 

Dunnett, V. (1955) A Multiple Comparison Procedure for Comparing Several 

Treatments vith a Control. JASA 50, 1096-1211. 

Heindel, J.J., Gulati, D.K., Mounce, R.C , Russell, S.R., and Lamb, J.C, IV. 

(1989) Reproductive Toxicity of Three Phthalic Acid Esters in a 

Continuous Breeding Protocol. Fund, and Appl. Toxicol, (in press). 

Jonckheere, A.R. (1954) A Distribution-free K-sample Test Against Ordered 

Alternatives. Biometrika 41, 133-145. 

Kruskal, V.H. and Vallis, V.A. (1952) Use of Ranks in One-criterion Variance 

Analysis. JASA 47, 583-621. 

Lamb, J.C, IV, Chapin, R.E., Teague, J., Lavton, A.D., and Reel, J.R. (1987) 

Reproductive Toxicity of Four Phthalate Acid Esters: Structure Activity 

Relationships. Toxicol. Appl. Pharmacol. 88, 255-269. 

Lamb, J.C, IV and Chapin, R.E. (1985) Experimental Models of Male 

Reproductive Toxicology. In: Endocrine Toxicology, J.A. Thomas, K.S. 

Korach and J.A. McLachlan, eds, Raven Press, Nev York, NY, pp 85-115. 
P.49 

45 

Lamb, J.C, IV, Maronpot, R.R., Gulati, D.K., Russell, V.S., Hommel-Barnes, 

L., and Sabharval, P.S. (1985) Reproductive and Developmental Toxicity 

of Ethylene Glycol in the Mouse. Toxicol. Appl. Pharmacol. 81, 100-112. 

Morrison, D.F. (1976) Multivariate Statistical Methods. McGraw Hill, Nev 

York. 

Morrissey, R.E., Collins, J.J., Lamb, J.C, IV, Manus, A.G., and Gulati, D.K. 

(1988) Reproductive Effects of Theophylline in Mice and Rats. Fund, and 

Appl. Toxicol. 10, 525-536. 

National Technical Information Service, U.S. Department of Commerce, 5285 Port 

Royal Road, Springfield, VA 22161 (703-487-4650). 

Neter, J. and Vasserman, V. (1974) Applied Linear Statistical Models. 

Richard Irvin, Inc., Homevood, IL. 

Shirley, E. (1977) A Non-Parametric Equivalent of Williams's Test for 

Contrasting Increasing Dose Levels of a Treatment. Biometrics 33, 

386-389. 

Soares, E.R., Sheridan, V., Haseman, J.K., and Segal, M. (1979) Increased 

Frequencies of Aberrant Sperm as Indicators of Mutagenic Damage in Mice. 

Mutation Res. 64. 27*35. 

Williams, D.A. (196%) A HgUt on Shirley's Nonparametric Test for Comparing 

Several Dose Levels with a Zero-Dose Control. Biometrics 42, 183-186. 

Vyrobek, A.J. and Bruce, V.R. (1975) Chemical Induction of Sperm 

Abnormalities in Mice. Proc. National Academy of Sciences, USA, 72, 

4425-4429.