National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 1 

February 1S76 

GUIDELINES FOR 

CARCINOGEN BIOASSAY 

IN SMALL RODENTS 

NCI-CG-TR-1 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 
NCI-CG-TR-1

 

February, 1976 

GUIDELINES FOR 

CARCINOGEN BIOASSAY 

IN SMALL RODENTS 

James M. Sontag, Ph. D. 

Manager, Bioassay Operations Segment 

Norbert P. Page, D. V. M. 

Chief, Carcinogen Bioassay and Program Resources Branch 

Umberto Saffiotti, M. D. 

Associate Director, Carcinogenesis Program and 

Chief, Experimental Pathology Branch 

CARCINOGENESIS PROGRAM 

DIVISION OF CANCER CAUSE & PREVENTION 

NATIONAL CANCER INSTITUTE 

BETHESDA, MARYLAN D 

DHEW Publication No. (NIH) 76-801 
For sale by the Superintendent of Documents, U.S. Government Printing Office

 

Washington, D.C. 20402 - Price $1.80

 

Stock Number 017-042-00118-8 
PREFACE
 

The National Cancer Act of 1971 provides legislative authority for the
 

National Cancer Institute to plan and develop an expanded, intensified,
 and coordinated cancer research program. An urgent and essential part
 

of this program is the need to protect the public from chemical and
 

physical carcinogenic hazards and their effects. In an effort to meet
 

this need, the Carcinogenesis Program of the Division of Cancer Cause
 

and Prevention has undertaken as one of its major activities the
 

identification of carcinogens in the environment and workplace. The
 

responsibility to develop and maintain this activity has been assigned
 

to the Bioassay Operations Segment in conjunction with the Carcinogen
 

Bioassay and Program Resources Branch.
 

It is the purpose of this document to provide guidelines for the
 

bioassay of chemicals for carcinogenic potential in small rodents. The
 

initial draft of this document was written following a workshop at
 

which the Carcinogenesis Bioassay Program's protocols were reviewed by
 

experts in the various scientific disciplines applicable to carcinogen
 

bioassay. The recommendations made at the workshop were reviewed by
 

investigators active in the NCI Bioassay Collaborative Program. Later
 

draft versions were reviewed by members of the scientific community at
 

large. Included among the latter were workers affiliated with
 

universities, other Federal agencies, commercial organizations, and
 

trade associations, as well as ones from institutes and laboratories
 

located abroad. An attempt was made to incorporate as part of this
 

document as many of the reviewers' comments as were compatible with its
 

intent. As a result of this extensive review by scientists
 

representing diverse interests in our society, it is hoped that these
 

guidelines will serve as the basis to standardize those aspects of a
 

large-scale carcinogen bioassay which are essential to its scientific
 

acceptability.
 
Appreciation is expressed to the individuals who participated in the
 

Workshop on Carcinogen Bioassay Protocols held November, 1973, at the
 

National Cancer Institute, Bethesda, Maryland. They were:
 

Richard R. Bates, National Cancer Institute
 

Thomas P. Cameron, National Cancer Institute
 

Kenneth Davis, National Center for Toxicological
 

Research
 

Thomas R. Fears, National Cancer Institute
 

Leo Friedman*, Food and Drug Administration
 

Harold C. Grice, Canadian Food and Drug Directorate
 

Herman F. Kraybill, National Cancer Institute
 

Norbert P. Page, National Cancer Institute
 

Umberto Saffiotti, National Cancer Institute
 

James M. Sontag, Chairman, National Cancer Institute
 

Robert A. Squire, National Cancer Institute
 

Borge M. Ulland, Litton Bionetics (Frederick Cancer
 

Research Center)
 

Elizabeth K. Weisburger, National Cancer Institute
 

*Deceased
 

We also express our gratitude to the many individuals who have reviewed
 

the material contained in this document and provided us with their
 

comments. These comments have added greatly to the overall quality of
 

the guidelines. In addition, we thank the many NCI staff members who
 

have labored not only over difficult concepts but also over the
 

sometimes more difficult task of putting them into clear and meaningful
 

statements. In this regard, special recognition is given to Dr.
 Cipriano Cueto.
 

Questions or comments regarding the material contained in this document
 

may be addressed to: Dr. James M. Sontag, Manager, Bioassay
 

Operations Segment, Landow Building, Room A-306, National Cancer
 

Institute, Bethesda, Maryland, 20014.
 
TABLE OF CONTENTS 

page 

I. Introduction 1 

II. General 2 

III. Animal Husbandry 3
 

A. Source 3
 

B. Distribution 3
 

C. Quarantine 3
 

D. Strain/Species 4
 

E. Animal Facilities 4
 

1. Physical Plant 4
 

2. Construction and Materials 4
 

3. Animal Rooms 5
 

4. Ventilation, Temperature and Humidity 5
 

5. Emergency Power 5
 

F. Animal Identification 5
 

G. Cages/Racks 6
 

H. Filters 6
 

I. Feeders 6
 

J. Water/Water Bottles 6
 

K. Rations 7
 

L. Bedding 7
 

IV. Safety and Health 8
 

A. Personnel Protection 8
 

B. Animal Protection 8
 

C. Protective Clothing and Equipment 8
 

V. Chemical Quality and Treatment Mixture Control 9
 

A. Storage/Shelf Life 9
 

B. Purity 9
 

C. Stability 9
 

D. Homogeneity/Concentration 9
 

E. Treatment Mixtures 9
 

VI. Animal Randomization 10
 

A. Randomization Procedure 10
 

VII. Prechronic Toxic!ogy 11
 

A. Acute Toxicity Study 11
 
1. Need 11
 

2. Experimental Groups 11
 

3. Route 11
 

4. Dose Levels 11
 

5. Treatment/Observation Periods 11
 

6. Observations 12
 

7. Dose Selection 12
 

B. Repeated-Dose Study 12
 

1. Need 12
 

2. Experimental Groups 12
 

3. Route 12
 

4. Dose Levels 12
 

5. Treatment/Observations Periods 13
 

6. Observations 13
 

7. Dose Selection 13
 

C. Subchronic Study 13
 

1. Need 13
 

2. Experimental Groups 13
 

3. Route 13
 

4. Dose Levels 14
 

5. Treatment/Observation Periods 14
 

6. Observations 14
 

7. MTD Determination 14
 

VIII. Chronic Study 16
 

A. Need 16
 

B. Experimental Groups 16
 

1. Animal Groups 16
 

2. Animal Age/Group Size 17
 

C. Route 17
 

D. Dose Levels 18
 

E. Treatment Period 18
 

F. Observation Period 18
 

G. Observations 19
 

H. Termination 19
 

IX. Carcinogen Treatment Group 20
 

A. Need 20
 

B. Carcinogen/Dose Level 20
 

C. Animals/Group Size 20
 

X. Data Records and Reports 21
 

A. Required Information 21
 

B. Methods of Collection 21
 
1.	 Logbooks 21
 

2.	 Carcinogenesis Bioassay Data 21
 

System (CBDS)
 

C.	 Reports 22
 

1.	 Chemicals On Carcinogenesis 22
 

Bioassay
 

2.	 Bioassay Information Sheet 29
 

3.	 Technical Report 29
 

4.	 Open Reports 29
 

APPENDICES
 

A.	 Animal Cage Weight-Space Specifications 31
 

B.	 NIH Open Formula Rat and Mouse Ration 32
 

C.	 Safety Standards for Research Involving Chemical 37
 

Carcinogens
 

D.	 Animal Randomization 50
 

E.	 Bioassay Pathology Procedures 51
 

F. Carcinogen Bioassay Information 58
 

General References 64
 

I. INTRODUCTION
 

Most human cancers are believed to be caused by exposure to extrinsic
 

factors, among which chemical agents are thought to be a major
 

contributor. These agents must be identified,, evaluated, and
 

controlled if the incidence of human cancer is to be reduced. For this
 

reason and the fact that many of these chemicals may have great social
 

and economic impact, it is essential that the procedures used to
 

determine their carcinogenicity be established on the best scientific
 

bases as are practically possible. However, differences in scientific
 

approaches and endpoints, as well as economic considerations, exclude
 

the use of any single set of procedures to meet the objectives of all
 

carcinogen bioassay studies. Notwithstanding these differences,
 certain features are common to all well designed and properly conducted
 

long-term animal studies.
 

The guidelines contained herein are used by the NCI CARCINOGENESIS
 

BIOASSAY PROGRAM (CBP)^ to screen environmental and occupational
 

chemicals for carcinogenicity by their oral administration to small
 

rodents. However, they also may be applicable, in part or in toto, to
 

other long-term animal studies that have different procedures,
 objectives, or endpoints. It is not the intent of this document to
 

address these variations. Although there are no substitutes for good
 

animal care practices, deviations from these guidelines may sometimes
 

be necessary for those laboratories not specially equipped to conduct
 

large-scale bioassay studies.
 
- 2 ­

II. GENERAL
 

The recommendations outlined in the Guide for the Care and Use of
 

Laboratory Animals1 should be adhered to throughout the bioassay
 

study. The general areas covered are: 1. Laboratory Animal
 

Management, 2. Laboratory Animal Quality and Health, 3. Personnel, 4.
 Use of Laboratory Animals, and 5. Physical Plant. When more rigid or
 

specific standards should be applied they are noted in the appropriate
 

sections of this document.
 

1. Prepared by the Committee on Revision of the Guide for Laboratory
 

Animal Facilities and Care of the Institute of Laboratory Animal
 

Resources, National Research Council, DHEW Pub. No. (NIH) 74-23, Super­ intendent of Documents, U.S. G.P.O., Washington, D.C., 20402.
 
- 3 ­

III. ANIMAL HUSBANDRY
 

III.A. Source Although it is ideal to have an animal production
 

colony associated with a large-scale bioassay operation, animals
 

supplied from commercial stocks are acceptable, providing they are
 

high quality, disease free, genetically stable, and adequately
 

identified as to colony source. Animals should be shipped in
 

sturdy, compartmented, and filtered containers. Arrangements should
 

be made with the carrier to assure rapid and optimal shipping
 

conditions that minimize trauma and extremes in temperature. Prompt
 

notification of the animals' arrival should be given by the carrier
 

to the receiving laboratory. Animals should be picked up
 

immediately and placed into quarantine.
 

The animal strain/species used to study a test agent and its
 

appropriate controls should be supplied from the same source. When
 

animals from different sources are used to study different test
 

agents, they should not be mingled and, optimally, should be
 

maintained in separate rooms.
 

III.B. Distribution During the quarantine period and acute
 

toxicity and repeated-dose studies, animals may be caged together
 

according to the weight-space specifications (Appendix A)
 

recommended in the Guide for the Care and Use of Laboratory Animals.
 

However, for the subchronic and chronic studies, animals should be
 

distributed from the outset of the studies as if they were in the
 

upper weight range. This will obviate the need to later
 

redistribute them to remain within the weight-space specifications.
 Even though the space to weight specification may be exceeded during
 

the subchronic and chronic studies, no cage should contain more than
 

five animals. As animals die or are sacrificed, surviving animals
 

should not be combined or redistributed among the cages.
 

III.C. Quarantine The quarantine area should be physically
 

separated from the testing area. Newly arrived animals should be
 

taken, in their unopened shipping containers, directly to the
 

quarantine area. A separate quarantine area should be provided for
 

each species. Animals that are unsuitable by reason of size,
 health, or other criterion should be immediately discarded. Animals
 

should be quarantined a minimum of seven days, after which they
 

2. Most mice and rats now used come from production colonies developed
 

and monitored by the CBP. These are periodically restarted with
 

breeders obtained from the NIH Division of Research Services.
 
- 4 ­

should be reexamined and those unsuitable should be discarded. A
 

small, randomly selected number of animals from each shipment should
 

be sacrificed and examined for parasites, enteric pathogens, and
 

other diseased conditions.3 When an epizootic disease is found
 

among the animals, the entire shipment from which they came should
 

be discarded. The quarantine area should be disinfected prior to
 

the receipt of additional animals.
 

III.D. Strain/'Species Both sexes of at least two species should
 

be used for the bioassay of each test agent."4 Considerations in
 

selecting the proper species and strains should include the
 

spontaneous tumor incidence, sensitivity to tumor induction,
 availability, genetic stability, hardiness, and longevity.
 

III.E. Animal Facilities The physical design and maintenance of
 

the animal facilities are of great importance in assuring high
 

standards of animal care, chemical and biological hazard control,
 and the proper conduct of the bioassay studies.
 

III.E.I. Physical Plant The animal facilities should be
 

designed and operated to minimize the introduction of external
 

biological and chemical agents into the building, as well as
 

between individual animal rooms. The animal facilities should
 

be effectively separated from the offices, laboratories, and
 

other rooms that are not essential to the maintenance or
 

treatment of the animals. The "clean-dirty" corridor flow
 

should be used to minimize the inadvertent transfer of
 

contaminants into the animal rooms. Service areas should be
 

located to effectively support the animal facilities.
 

III.E.2. Construction and Materials The construction of the
 

building and the materials used should facilitate the sanitation
 

of the bioassay operation. Utilities such as water and electric
 

lines and ventilator ducts should be located outside of the
 

animal rooms. Components that interrupt the surface integrity
 

3. See Guide to Infectious Diseases of Mice and Rats. A report of the
 

Committee on Laboratory Diseases, Inst. Lab. Animal Resources, NRC,
 NAS, 2101 Constitution Ave., Washington, D. C., 20418; Pub. ISBN
 

0-309-01914-1.
 

4. The CBP uses primarily the B6C3F! hybrid (C57BL/6 x C3H/f) mouse
 

and either the Fischer strain 344 or Osborne-Mendel rat.
 
-- 5 -­

of a room, e.g., electrical outlets and light fixtures, should
 

be recessed into the surface and sealed. Seamless materials
 

that are durable, waterproof, and fire-resistant should be used
 

for interior surfaces. Paint and glazes should be highly
 

resistant to chemical solvents, scrubbing, high-pressure sprays,
 and impact. Floor drains and windows are not necessary in
 

facilities housing small rodents. Where they already exist,
 they should be blocked and sealed.
 

III.E.3. Animal Rooms The size and number of animal rooms
 

should be sufficient to assure a separate room for each species.
 Ideally, a separate room also should be used for each test agent
 

under study. Cages with solid sides and bottoms and covered by
 

filter tops must always be used when studies cannot be
 

physically separated.
 

11 I.E.4. Ventilation, Temperature^ and Humidity Special
 

attention should be given to ensure that the animal facilities
 

are properly ventilated. Each animal room should undergo 10-15
 

fresh-air changes per hour. The air pressure should be adjusted
 

so that the animal rooms are slightly positive to the "dirty"
 

corridor and negative to the "clean" one. All air must be
 

adequately filtered before it enters or leaves the animal
 

facilities. The temperature and humidity should be maintained
 

at those settings that have been reported to be optimal for the
 

animal species being used.5 An automatic recording and alert
 

system should be used to monitor the ambient conditions in each
 

animal room.
 

11 I.E.5. Emergenoy Power An emergency power source should be
 

available as a backup to the primary system. Its generating
 

capacity should be sufficient to power the animal facilities'
 

air-condition and light systems.
 

III.F. Animal Identification Whenever individual animal data are
 

to be routinely recorded, each animal should be marked at the outset
 

of the study by a standard method of identification; e.g., ear
 

notching, toe clipping, or tagging.
 

5. The CBP recommends a temperature of 74°F ±2°F (23.3°C ±1.1°C) and a
 

relative humidity of 40% ±5% be maintained in rat and mouse rooms.
 
- 6 ­

III.G. Cages/Racks Plastic or stainless-steel cages with solid
 

sides and bottoms should be used, especially for studies in which
 

the test agent may be scattered or excreted. Wire-mesh cages may be
 

required for certain types of studies; e.g., inhalation. Animals
 

housed in cages with solid sides and bottoms should be changed to a
 

sanitized cage6 with fresh bedding as frequently as necessary, but
 

not less than once weekly. Wire-mesh cages should be sanitized no
 

less than once every two weeks.
 

Racks may be of either the shelf or suspended-drawer type and should
 

be sanitized either in place or moved to a wash area for cleaning.
 If a rack washer is not available, they should be scrubbed with a
 

suitable detergent and hosed down under high pressure. Racks should
 

be sanitized at least once every other week.
 

III.H. Filters When cages with solid sides and bottoms are used,
 nonwoven polyester fiber filters should be placed over them as a
 

disease control and chemical containment measure. Bonnets or filter
 

sheets should be replaced by sanitized ones at least once every two
 

weeks.
 

III.I. Feeders Feeders that are adequately designed to prevent
 

soiling, bridging, and scattering of the feed are acceptable when
 

pellet-type rations are used. Although no feeder is completely
 

satisfactory for meal feed, a hopper-type feeder that is firmly
 

attached to the cage appears to cause the least problems. However,
 this type of feeder may still require daily "bumping" to dislodge
 

bridged meal. An open, unfixed feed cup should not be used nor
 

should the feed be placed directly onto the cage floor. A sanitized
 

feeder6 should be supplied at least once weekly.
 

III.J. Water/Water Bottles An adequate supply of fresh and
 

suitably treated water should be provided ad libitum. Potential
 

pathogens carried in the water should be killed or removed through
 

appropriate treatment; e.g., sterilization, pasteurization, or
 

filtration. Each cage should be supplied at least twice weekly with
 

a sanitized water bottle, stopper, and sipper tube.6 The bottles
 

should be filled and the stoppers and sipper tubes inserted into
 

them only outside of the animal rooms. Empty or partially full
 

6. To assure destruction of pathogens, water at 180°F (82°C) should be
 

used for a period in the rinse cycle. Sanitization of bottle stoppers
 

and sipper tubes also may be done by either germicide treatment before
 

washing or by boiling after washing.
 
- 7 ­

water bottles should be replaced rather than refilled. When an
 

automatic watering system is used, the valve-end should be located
 

in such a manner as to prevent accidental flooding of the cage.7
 

III.K. Rat-ions A nutritionally balanced standard laboratory feed
 

which supports normal growth and maintenance should be used.8
 

Other factors to consider in choosing a feed should include the
 

constancy of its major ingredients and their sources, its moisture
 

content, freshness, storage characteristics, and timely delivery.
 Non-nutritional intentional additives, such as antibiotics and
 

estrogens, should be avoided. Whenever a change in diet manufacture
 

occurs during the course of a study, both the test and associated
 

control groups should be switched at the same time to the new feed.
 Periodic analyses of the feed for pesticide (chlorinated
 

hydrocarbons, etc.), mycotoxin (aflatoxins, etc.), and industrial
 

(polychlorinated biphenyls, etc.) contaminants are recommended. The
 

data from such analyses should be retained and included in the final
 

report on each test agent. Whenever practical and consistent with
 

the disease control program, the feed should be sterilized. Care
 

should be taken that the nutrients are not degradated or the
 

palatability of the feed altered. Feed should be provided as often
 

as necessary, but not less than once weekly, to assure an adequate
 

supply of fresh rations. When a test agent is given in the diet,
 its stability also must be considered as a factor in determining how
 

often the feed should be renewed (see Section V.C.). Contaminated
 

or spoiled feed should be replaced within the same working day in
 

which it is found.
 

I ILL. Bedding Although mycotoxin-free ground corncob may be
 

used, heat-treated hardwood chips are considered the most desirable
 

type of bedding. Softwood chips or creosoted wood should not be
 

used. The bedding should be sterilized. In studies in which open
 

wire-mesh cages are used, an absorbent material that effectively
 

collects and holds waste matter should be placed under them.
 

7. A break in the primary containment barrier is created by the
 

aperature in the cage which allows the animals to reach the valve end.
 Thus, before use of an automatic: watering system, consideration should
 

be given to the nature of the compounds to be tested and safety
 

requirements.
 

8. Consideration should be given to the use of the NIH Open Formula
 

Rat and Mouse Ration (Appendix B).
 
- 8 ­

IV. SAFETY and HEALTH
 

IV.A. Personnel Protection Each test agent should be treated as a
 

potential carcinogen. Thus, every precaution should be taken to
 

prevent inadvertent exposure of personnel and the environment to the
 

test agent. Each aspect of the bioassay study, from receipt of the
 

test agent through histopathology, must be monitored and controlled.
 Personnel whose medical condition, e.g., depressed immune response,
 pregnancy, and steroid or cytotoxic drug treatment, may make them
 

unusually susceptible to the possible harmful effects of a test
 

agent should be excluded from any area where accidental exposure
 

might occur. Individuals who are allergic to laboratory animals
 

should not be exposed to them unless adequately protected and
 

approval has been given by the medical or safety officer. Each
 

laboratory should develop a safety and health plan for the handling
 

of potential carcinogens. Applicable federal, state, and local
 

regulations must be adhered to, as well as the National Cancer
 

Institute's safety standards for research involving chemical
 

carcinogens (Appendix C).
 

IV.B. Animal Protection Strict hygienic and disease prevention
 

measures should be routinely practiced. Access to the animal
 

facilities should be restricted to only those individuals essential
 

to their operation. Personnel should receive adequate animal care
 

and personal hygiene training and instruction as to the proper
 

operating procedures. Personnel with respiratory infections or
 

other disease conditions which may affect the animals' health should
 

be excluded from the animal rooms.
 

IV.C. Protective Clothing and Equipment All personnel who may be
 

exposed to either the animals or test agents must be adequately
 

protected. A complete change of clean clothing should be provided
 

daily and should include a fully fastened laboratory suit, gloves,
 boots, and head cover. The protective clothing should not be worn
 

outside of the work area. An appropriate face mask or respirator
 

should be worn as protection against dust, mists, or fumes.
 
- 9 ­

V. CHEMICAL QUALITY and TREATMENT MIXTURE CONTROL
 

V.A. Storage/Shelf Life The proper method(s) for the long-term
 

storage of each bulk test agent should be determined prior to its
 

bioassay. A sample of the bulk test agent should be analyzed
 

periodically during the study to ensure that it has retained its
 

original characteristics.
 

V.B. Purity The purity of each test agent should be determined
 

prior to its bioassay. In some instances, identification and
 

determination of the percent of each impurity, as well as the
 

purification of the test agent, may be desirable.
 

V.C. stability The; stability of each test agent should be
 

determined under the same conditions in which it will be
 

administered, e.g., in the feed and/or vehicle, and temperature
 

and/or pH, prior to the start of the bioassay study. This analysis
 

should be done on samples taken from newly prepared treatment
 

mixtures and from the last of usable ones; e.g., diet mixes. The
 

frequency at which fresh treatment mixtures are prepared may depend
 

on the stability of the test agent under its conditions of storage
 

and/or administration.
 

V.D. Homogeneity/Concentration When the test agent is incorporated
 

into the feed, its homogeneity and concentration in the diet mix
 

should be determined before the start of the bioassay study. Random
 

samples from freshly mixed batches should be analyzed periodically
 

during the study to ensure that the proper mixing and formulation
 

procedures are being used.
 

V.E. Treatment Mixtures Treatment mixtures should be stored in
 

inert, shatter-proof containers with air-tight covers. Each
 

container should be clearly marked so that its contents can be
 

easily and accurately identified. The storage containers should be
 

 basis. A record also should be kept of the preparation and
 

located in a secure area that is free of

restricted access. congestion and with 

An inventory of each treatment mixture should be maintained on a 

current


usage of each treatment mixture, including the dates and quantities
 

prepared or used and the names of the responsible individuals.
 
- 10 ­

VI. ANIMAL RANDOMIZATION
 

VI.A. Randomization Procedure Ideally, all animals should be
 

within 2-3 days of the same age. The animals should be initially
 

segregated into equal weight distribution groups and then divided
 

into experimental groups (treatment and appropriate controls) using
 

formal randomization methods (Appendix D). This procedure will
 

ensure that each group will contain animals of approximately the
 

same weight and age.
 
- 11 ­

VII. PRECHRONIC TOXICOLOGY
 

The purpose of the prechrom'c toxicologic studies is to predict the
 

maximum tolerated dose (MTD) of the test agent that can be given to the
 

animals during the chronic study. This dose is predicted on the basis
 

of the data collected from a 90-day subchronic study. However, when
 

there are insufficient toxicologic data on the test agent, an acute
 

and/or short-term repeated dose study may be needed prior to the
 

subchronic study.
 

VILA. Acute Toxicity Study
 

VII.A.I. Need An acute toxicity study may be necessary when
 

data are unavailable as to the toxicity of the test agent. Its
 

purpose is to determine the lethality of the test agent as well
 

as to gain other information on its acute toxicity. The data
 

collected should be used as a guideline to determine the dose
 

range to be used in subsequent studies designed to predict the
 

MTD.
 

VII.A.2. Experimental Groups To determine the lethality of
 

the test agent, at least five animals (no older than young
 

adults) of each sex and strain to be studied in the chronic
 

phase should be used at each of the dose levels to be tested.
 

VII.A.3. Route The route of administration of the test agent
 

should be the same one to be used in the chronic study.
 

VII.A.4. Dose Levels The lethality of the test agent can be
 

estimated by use of a series of three of more dose levels which
 

increase in a geometric progression; e.g., by a factor of two.
 For the purpose of this study, a precise lethal dose (LD) value
 

need not be determined.
 

VII.A.5. Treatment/Observation Periods On day one of the
 

study the animals should be administered an acute dose of the
 

test agent. The duration of the exposure may be either short,
 e.g., a single injection or intubation, or continuous such as in
 

the feed or water. Whatever the route or mode of
 

administration, the duration of exposure should not exceed 24
 

hours. The test agent should be given in the same formulation
 

or closely approximate the one to be used in the chronic study.
 
- 12 ­

After treatment, the animals should be held a minimum of 14 days
 

for observation.
 

VII.A.6. Observations It is important that all relevant
 

clinical signs be recorded, including animal weights at the
 

beginning and termination of the study and the time to death of
 

each animal. An indication as to the type of lesions produced
 

by the test agent may be obtained by necropsies on some or all
 

of the animals.
 

VII.A.7. Dose Selection An analysis of the data should be
 

made to estimate the dose at which 10% of the animals are
 

killed. This dose can be found by a probit or logit analysis of
 

the data plotted on appropriate graph paper. The dose selected
 

should be the highest one used in subsequent studies designed to
 

predict the MTD.
 

VII.B. Repeated-Dose Study
 

VII.B.I. Need The 14-day repeated-dose study should be done
 

when the available toxicologic data are insufficient to
 

determine the dose levels to be used in the subchronic study.
 

VII.B.2. Experimental Groups Five animals (no older than
 

young adults) of each sex and strain to be studied in the
 

chronic phase should be used at each of the dose levels to be
 

tested. A similar number of comparable animals should be
 

used as controls.
 

VII.B.3. Route Administration of the test agent should be by
 

the same route to be used in the chronic study.
 

VII.B.4. Dose Levels Five dose levels should be initially
 

tested. The upper level should be the one that is estimated to
 

produce no more than 10% lethality following administration of a
 

single acute dose of the test agent. The remaining levels
 

should be fractions thereof; e.g., one-half, one-fourth,
 one-eighth, etc. If these fail to provide the information
 

needed to select the dose range to be used in the subchronic
 

phase, the study should be extended using the initial results to
 

determine whether higher or lower doses should be further
 

tested.
 
- 13 ­

VII.B.5. Treatment/Observation Periods Animals should be
 

treated daily with the test agent for a period not exceeding 14
 

days, after which they should be held another 24 hours before
 

sacrifice. The exposure may be of either a short duration,
 e.g., a single daily injection or intubation., or continuous such
 

as in the feed or water. The test agent should be given in the
 

same formulation as the one to be used in the chronic study.
 

VII.B.6. Observations Daily observations should be made for
 

clinical signs of toxic effects. Weights should be recorded at
 

the beginning of the study and thereafter at weekly intervals.
 For some test agents, especially those that may produce delayed
 

toxicity or cumulative effects, other measurements, e.g., organ
 

function, body burden, absorption, and excretion also may
 

provide information which is useful in predicting the proper
 

subchronic dose levels. Animals should be necropsied and
 

pathologic studies done in accordance with the Bioassay
 

Pathology Procedures (Appendix E).
 

VII.B.7. Dose Selection A review of the data should be made
 

to select the maximum dose to be used in the subchronic study.
 This dose should be the highest one that did not produce
 

clinical signs of toxicity, pathologic lesions, or mortality.
 

V11.C. Subehronia Study
 

VII.C.I. Need The puroose of the subchronic study is to
 

predict the toxic effects which may occur in the animals during
 

the chronic administration of the test agent. Based on the
 

subchronic observations, a prediction is made of the maximum
 

tolerated dose (MTD) that can be given to the animals in the
 

chronic study without producing unwanted side effects.
 

VII.C.2. Experimental Groups The experimental groups
 

composing the subchronic study should include the test and
 

appropriate control animals of each sex and strain to be used in
 

the chronic study. Each experimental group should contain 10
 

animals of one sex and strain at a single dose level. The age
 

of the animals should be the same as those to be used to start
 

the chronic study.
 

VII.C.3. Route Administration of the test agent should be by
 

the same route to be used in the chronic study.
 
- 14 ­

VII.C.4. Dose Levels Five dose levels should be tested — the
 

highest being the one that does not produce clinical signs of
 

toxicity, pathologic lesions, or mortality in short-term acute
 

or repeated-dose studies. The remaining dose levels tested
 

should be fractions thereof; e.g., one-half, one-fourth,
 one-eighth, etc. If these fail to bracket the range in which
 

the MTD can be predicted, the study should be extended and the
 

information from the initial attempt used as a guide in setting
 

the dose levels for the next series to be tested.
 

VII.C.5. Treatment/'Observation Periods The test agent should
 

be administered to the animals for 90 days9, according to the
 

same treatment schedule and formulation to be used in the
 

chronic study.10 The amount of test agent administered or
 

consumed by the animals should be quantitated. Although for
 

some modes of administration, e.g., in the feed, the intake
 

cannot be determined precisely, an estimate should be made of
 

the amount consumed.
 

VII.C.6. Observations Daily observations should be made for
 

clinical signs of toxic effects. For some test agents,
 especially those that may produce delayed toxicity or cumulative
 

effects, other measurements, e.g., organ function, body burden,
 absorption, and excretion, also may provide information which is
 

useful in predicting the proper chronic dose levels. Individual
 

animal weights should be recorded weekly and plotted so that the
 

weight gain of each group can be compared over the length of the
 

study. Animals should be necropsied and pathologic studies done
 

in accordance with the Bioassay Pathology Procedures (Appendix
 

E).
 

VII.C.7. MTD Determination A MAXIMUM TOLERATED DOSE (MTD)
 

should be selected for each sex of each strain to be used in the
 

chronic study. The MTD is defined as the highest dose of the
 

test agent given during the chronic study that can be predicted
 

not to alter the animals' normal longevity from effects other
 

than carcinogenicity. The MTD is estimated after a review of
 

the subchronic data. Since these data may not always be easily
 

9. For compounds that show delayed toxicity or cumulative effects, a
 

treatment period of 120-150 days or longer may be needed.
 

10. In some instances, a short observation period following treatment
 

may be desirable to predict the animals' recovery potential.
 
- 15 ­

interpretable, a degree of judgment is often necessary in
 

estimating the MTD. The MTD should be the highest dose that
 

causes no more than a 10% weight decrement11, as compared to
 

the appropriate control groups; and does not produce mortality,
 clinical signs of toxicity, or pathologic lesions (other than
 

those that may be related to a neoplastic response) that would
 

be predicted to shorten the animal's natural life span. Other
 

measurements (see VII.C.6.) also may be used to aid in
 

predicting the MTD.
 

11. Although a depressed weight gain is a clinical sign of toxicity,
 this particular effect is acceptable when estimating the MTD.
 
VIII. CHRONIC STUDY
 

VIII.A. Need The purpose of this study is to determine the
 

carcinogenicity of the test agent in both sexes of two species and
 

is designed to cover the greater part of the animals' life spans,
 

V111.B. Experimental Groups 

VI11.B.I. Animal Groups Each experimental group is composed
 

of animals of the same sex and strain receiving an identical
 

treatment. The usual experimental groups used in the chronic
 

study are shown below.
 

VIII.B.I.a. Test groups - Each test group is composed of
 

animals exposed to an identical dose level of the test
 

agent. For example, female strain X mice exposed to a
 

specific dose level would comprise one test group. Mice at
 

a different dose level or of another sex or strain would
 

comprise a separate test group.
 

VIII.B.l.b. Control groups - One or more different control
 

groups may be necessary to evaluate the carcinogenicity of
 

the test agent. The most common are:
 

VIII.B.l.b.i. Vehicle control groups - Animals in these
 

groups are treated with only the vehicle in which the
 

test agent is dissolved or dispersed.
 

VIII.B.l.b.ii. Untreated control groups - Animals in
 

these groups should be identical in every respect to
 

those in the other experimental groups except that they
 

receive no specific treatment.
 

Although the above controls may be adequate, the appropriate
 

matched control groups necessary to evaluate a test agent's
 

carcinogenicity also may be those receiving another material
 

or exposed to a given procedure used in its administration.
 Except for the specific treatment, all experimental groups
 

should be identical in every respect, including source and
 

conditions of maintenance.
 
- 17 ­

In addition to the appropriate matched controls, located in the
 

same room as the test groups, it also is desirable to have a
 

group of untreated animals (colony controls) in a separate room
 

where the likelihood of their inadvertent exposure to a test
 

agent is reduced. Colony control groups should be maintained
 

for each strain from each animal supplier. Ideally, colony
 

controls should be initiated whenever a new shipment of animals
 

is received.
 

VI11.B.2. Animal Age/Group Size At the start of the chronic
 

study, animals should be no older than 6 weeks of age and, if
 

possible, weanlings. Each test and appropriate control group
 

should contain 50 animals. When several test agents are placed
 

on study at the same time, common untreated and, where
 

applicable, other control groups may be used. The size of such
 

control groups should be increased by a factor equal to the
 

square root of the number of test agents to which they are
 

common. Thus, a control group common to 4 test agents would
 

contain 100 animals (100 = 50/4). Common control groups should
 

be located in the same animal room as the test groups are
 

housed.
 

VIII.C. Route Administration of the test agent should be by a
 

route that duplicates or closely approximates the one by which human
 

12
 exposure occurs.
 

12. When the route is oral, consideration should be given as to
 

whether the test agent should be administered by gastric intubation, in
 

a diet mix, or in the water. The introduction of the test agent by
 

gastric intubation offers greater hazard control, better quantisation,
 the need for less test agent, fresher preparations, easier storage, and
 

increased animal handling. Its disadvantages are that there is
 

generally less than maximum intake of the test agent, a solvent is
 

usually required, mortality may be increased, and animals need to be
 

closely matched by weight. Diet mix offers a greater total intake of
 

the test agent and may more closely simulate the mode of human
 

exposure. Its disadvantages are that contamination may be difficult to
 

control, the homogeneity of the mix is not assured, decomposition of
 

the test agent may occur during storage or while in the feeder, the
 

palatability of the feed may be affected, and the quantity ingested by
 

each animal may vary. The advantages and disadvantages of giving the
 

test agent in the water are similar to those encountered when the diet
 

mix is used as the mode of administration.
 
- 18 ­

VIII.D. Dose Levels At least two dose levels of the test agent
 

should be studied in each sex of each strain. The highest dose
 

should be the MTD, as predicted from the subchronic data, and the
 

remaining level(s) a fraction thereof.13 In instances in which the
 

test agent does not cause signs of toxicity at levels up to 5%
 

(50,000 ppm) in the diet, it is recommended that this concentration
 

not be exceeded except uncer special circumstances; e.g., when the
 

test agent is used as a major human dietary constituent. If
 

clinical signs of toxic effects appear during the course of the
 

chronic study, appropriate action must be taken to ensure maximum
 

animal survival. The type of action will depend upon the objective
 

of the study and may include lowering the dose, discontinuing
 

treatment, or changing the dose regimen. Although the doses may
 

need to be adjusted downward during the course of the study, they
 

should never be raised. Frequent and competent animal surveillance
 

must be constantly stressed.
 

VIII.E. Treatment Period The test agent should be given for a
 

sufficient time to produce a maximum response in the animals.
 Although prospectively this period can only be estimated, usually
 

the greater part of the animal's life span is considered
 

desirable.14 To reduce the possibility of exposing the necropsy
 

technicians to the test agent or its metabolite(s), treatment of the
 

animals should be discontinued one week prior to their scheduled
 

sacrifice date. Except when the test agent is given in the drinking
 

water or diet, the frequency of treatment will depend upon the route
 

of administration and mode of human exposure. When the test agent
 

is given in the water or in the feed as part of the diet mix, it
 

should be administered on a 7-day-a-week basis. The amount of test
 

agent administered or consumed by the animals should be quantitated.
 Although for some modes of administration, e.g., in the feed, the
 

intake cannot be determined precisely, an estimate should be made of
 

the amount consumed.
 

VIII.F. Observation Period It may be desirable to hold the
 

animals for an additional period after cessation of treatment. This
 

period of time may allow the tumors induced by the test agent to
 

13. The CBP usually uses the MTD and either the MTD/2 or MTD/4. Thus,
 when both sexes of two species are used, as many as 4 different MTD's
 

could be studied.
 

14. The CBP usually requires a minimum treatment period of 24 months.
 

At the end of this time an evaluation is made as to whether the
 

treatment period should be extended or terminated.
 
- 19 ­

show a biological potential, e.g., invasion, metastases, or
 

transplantability, that is not apparent at the end of the treatment
 

period. Also during this time other treatment-related lesions may
 

regress. An observation period of 3 to 6 months is usually
 

considered to be sufficient.
 

VIII.G. Observations High priority should be given to frequent
 

and competent animal surveillance. A viability check of every
 

animal should be made once in the morning and once in the late
 

afternoon on a 7-day-a-week basis. Animals in poor health or with
 

life-threatening tumors should be isolated. Those whose condition
 

makes it unlikely that they will survive another 24 hours should be
 

sacrificed. Every animal should be palpated and carefully examined
 

at least once a week.
 

Animal weights should be recorded on day one of the study and
 

thereafter at monthly intervals. Ideally, weights should be
 

recorded on an individual animal basis. The frequency of weighing
 

should be increased to once every two weeks when either a tumor is
 

detected in an animal from a test group or there is an apparent
 

deterioration in the animals' health. The same weighing schedule
 

used for the test-group animals also should be applied to their
 

matched controls. Feed consumption should be measured on a regular
 

basis. Usually such measurements can be conveniently made using the
 

same schedule used to record animal weights.
 

In certain cases, particularly for those chemicals that persist in
 

the animals, it 'may be possible and desirable to determine the
 

distribution and concentration of the test agent or its
 

metabolite(s) in the tissues, including blood. Such an analysis may
 

permit a correlation to be made between the tissue concentration and
 

the test agent's carcinogenicity.
 

VIII.H. Termination The animals in a test group should be
 

sacrificed when either the original or adjusted termination date is
 

reached.15 A test group should be terminated earlier only when its
 

cumulative mortality is sufficiently great so that prolongation of
 

the group is no longer statistically justified. Control group
 

animals should be sacrificed according to the original or adjusted
 

termination date, whichever is later. Animals should be necropsied
 

and pathologic studies done in accordance with the Bioassay
 

Pathology Procedures (Appendix E).
 

15. A test group is usually terminated when survival

the original number of animals in that group, reaches although

 10% of 

 in some 

circumstances it may be earlier or later. 
- 20 ­

IX. CARCINOGEN TREATMENT GROUP
 

IX.A. Need A Carcinogen Treatment Group (Positive Control) may be
 

desirable to determine whether the animals used to study the
 

carcinogenicity of the test agents respond in a predictable manner
 

to a known carcinogen. The results from a Carcinogen Treatment
 

Group also can be useful in evaluating the carcinogenicity of the
 

test agents. A Carcinogen Treatment Group need not be initiated
 

concurrently with each new set of test groups but should be started
 

at reasonable periodic intervals to monitor the animals'
 

responsiveness.
 

IX.B. Caroinogen/Dose Level Whenever possible the chemical
 

structure of the carcinogen should be similar to the class of test
 

agents under study. There also should be an adequate amount of
 

experimental data with the chosen carcinogen in the strains being
 

used so that the tumor type(s), latent period(s), and incidence(s)
 

can be reasonably predicted. The carcinogen should be of a
 

sufficient potency to produce a detectable number of tumors in the
 

exposed animals. Only a single dose level need be tested.
 

IX.C. Animals/Group size The sexes and strains of the animals
 

comprising the carcinogen treatment groups, and their conditions of
 

maintenance, should be identical to those used to study the
 

carcinogenicity of the test agents. Although fewer animals can be
 

used with potent carcinogens than with weaker ones, each treatment
 

group should contain at least 20 animals.
 
- 21 ­

X. DATA RECORDS and REPORTS
 

X.A. Required Information It is essential that all the
 

information and data pertaining to a bioassay study are completely
 

and accurately recorded on a current basis. This should include all
 

the information related to the test agent, animals, procedures, and
 

results. Because of its importance and the scope of the effort, a
 

plan for the collection of this information should be devised before
 

the start of a bioassay study. As a minimum, the information
 

recommended in the report by the Panel on Carcinogenicity Testing of
 

the International Union Against Cancer (Appendix Fj should be
 

collected and be available for review during and at the end of a
 

bioassay study.
 

X.B. Methods of Collection
 

X.B.I. Logbooks The key to a successful logbook system is
 

ease, accuracy, and completeness in recording and retrieving
 

data. However, when dealing with many studies and large numbers
 

of animals, manually maintained logbooks may become bulky and
 

difficult to manage. The preparation of reports requires the
 

extraction, review, consolidation, and tabulation of data from
 

the logbooks. Much of this time-consuming hand operation has
 

been overcome with the development, by the National Cancer
 

Institute, of a computerized system to collect, retrieve,
 tabulate, and analyze bioassay test data. This system is
 

briefly described in the sections below.
 

X.B.2. Carcinogenesis Bioassay Data System (CBDS) The CBDS
 

(Linhart, 1974) is used extensively by the CBP to collect,
 retrieve, tabulate, and analyze bioassay test data. It also
 

serves to manage and monitor the status and progress of
 

individual bioassay studies as well as to summarize the total
 

effort of the CBP. Data input is usually through a series of
 

forms (see pages 23 to 28) submitted to the CBP where they are
 

processed for entry into a computer. The Systematized
 

Nomenclature of Pathology (SNOP) is used to code the pathology
 

results collected on individual animals.
 

As the usefulness and integrity of the data output are only as
 

good as the accuracy of the data input, it is necessary to
 

closely monitor data before their entrance into the system. To
 

ensure that the data technicians at the field level have the
 

proper background, special training classes are periodically
 
- 22 ­

held at the NCI.16 After coding and prechecking, the data
 

forms are translated into a machine-readable format and entered
 

into the system, microfilmed, and stored as microfiche for
 

future reference. After computer editing for errors, the data
 

are available for selective or complete recall. Data output is
 

available as a series of standard or special reports and tables
 

presenting the data in the following ways:
 

a. bioassay studies underway within the total program or
 

within a particular contract effort,
 

b. an individual bioassay study, 

c. special pathology reports, 

d. survival and weight curves, and 

e. selected analysis of the test data. 

Many of the reports are generated for use by the field
 

investigator and other concerned individuals and may be readily
 

available. Other reports are for internal use or to support the
 

activities of other Governmental agencies and usually have a
 

limited distribution.
 

X.C. Reports In view of the possible use of bioassay test results
 

in establishing public policy,, the general release of such data is
 

normally done only after the data have been completely confirmed,
 verified (including pathology review), and statistically analyzed.
 The final publication is made through the issuance of a Technical
 

Report. Other reports of a less complete nature may precede this
 

final report.
 

X.C.I. Chemicals on Carcinogenesis Bioassay This report lists
 

all the chemicals under test for carcinogenicity by the CBP.17
 

16. For information regarding these classes or any phase of the data
 

system, write Carcinogenesis Bioassay Data system, P.O. Box I,
 Bethesda, Maryland 20014.
 

17. This report may be requested from the Chief, Carcinogen Bioassay
 

and Program Resources Branch, Landow Building, Room C319, National
 

Cancer Institute, Bethesda, Maryland 20014.
 
- 23 ­
- 24 ­
- 25 ­

- 27 ­
- 28 ­
- 29 ­

X.C.2. Bioassay Information Sheet This is an interim report
 

detailing the experimental design, expected completion dates,
 and other information relating to the bioassay of a particular
 

chemical. Preliminary results or conclusions are not included
 

unless they appear to be unequivocal.
 

X.C.3. Technical Report The results from each bioassay study
 

of a particular chemical are fully documented in this report.
 It contains all of the information recommended in the report by
 

the Panel on Carcinogenicity Testing of the International Union
 

Against Cancer (Appendix F) and is prepared jointly by the field
 

investigator and the NCI staff. The results are tabulated in a
 

chronological sequence so that each animal is easily followed.
 This includes time and mode of death and the associated
 

histopathology. In addition to the "raw" data, the Technical
 

Report contains a narrative of the experimental design, conduct
 

of the study, pathology results, summary tables of the data,
 statistical analyses, discussion, and conclusions. The
 

Technical Report provides a full documentation on the bioassay
 

study of a particular test agent and is available to the public.
 

X.C.4. Open Reports These reports usually take the form of
 

journal articles or papers presented at scientific meetings.
 After the bioassay test results have been appropriately
 

analyzed, evaluated, and approved, the CBP encourages the
 

dissemination of such reports on every chemical tested under its
 

aegis.
 

- 31 ­

APPENDIX A
 

ANIMAL CAGE
 

WEIGHT-SPACE SPECIFICATIONS
 

The weight-space specifications, shown below, conform with those
 

recommended in the Guide for the Care and Use of Laboratory Animals.
 

SPECIES WEIGHT FLOOR AREA/ANIMAL HEIGHT*
 

(SQUARE)
 

(gm) (cm) (in) (cm) (in)
 

Mouse Up to 10 

10-15 

16-25 

Over 25 

39 

52 

77 

97 

( 6) 

! 8! ( 12 

( 15) 

12.7 

12.7 

12.7 

12.7 

(5) 

(5) 

(5) 

(5) 

Rat Up to 100 

100-200 

201-300 

Over 300 

110 

148 

187 

258 

( 17)

23 

( 29) 

( 40) 

17.8 

17.8 

17.8 

17.8 

!7

(7) !

(7) 

(7) 

Hamster Up to 60 

60-80 

81-100 

64.5 

83.9 

103.2 

!10! ( 13) 

16 

15.2 

15.2 

15.2 

(6)

(6) 

6 

Over 100 122.6 ( 19) 15.2 (6) 

Guinea 

pig 

Up to 250 

250-350 

Over 350 

277 

374 

652 

( 43) 

( 58 

(101) 

17.8 

17.8 

17.8 

(7) 

(7) 

(7) 

*Height means from the resting floor to the cage top.
 
- 32 ­

APPENDIX B
 

NIH OPEN FORMULA RAT AND MOUSE RATION
 

This specification (adapted from NIH-ll-133c, Jan. 2, 1973) is for an
 

open formula laboratory rat and mouse ration which is void of any feed
 

additives containing antibiotics or estrogenic activity. It should be
 

noted that the optimum balance of dietary constituents for animals in
 

carcinogen bioassay studies has not been clearly defined. Thus,
 deviations from this specification may sometimes be acceptable.
 

Materials - Material shall be as specified below:
 

Ingredient Percentage by Weight
 

Dried skim milk 5.00
 

Fish meal (60% protein) 10.00
 

Soybean meal (49% protein) 12.00
 

Alfalfa meal (dehydrated 17% protein) 4.00
 

Corn gluten meal (60% protein) 3.00
 

Ground #2 yellow shelled corn 24.50
 

Ground hard winter wheat 23.00
 

Wheat middlings 10.00
 

Brewer's dried yeast 2.00
 

Dry molasses 1.50
 

Soy oil 2.50
 

Salt 0.50
 

Dicalcium phosphate 1.25
 

Ground limestone 0.50
 

Pre-mixes 0.25
 

100.00
 

Ingredients should be ground to pass through a U.S. Standard Screen
 

No. 16 before mixing.
 
- 33 ­

Vitamin Fortification Per Ton (2,000 Ibs.) of Finished Product:
 

Vitamin Amount 

A 5,500,000.0 I.U. 

Ds 4,600,000.0 I.U. 

K
 

A-tocopheryl acetate
 

Choline
 

Folic acid
 

Niacin
 

d Pantothenic acid
 

Riboflavin
 

Thiamine
 

812
 

Pyridoxine
 

Biotin
 

Mineral Fortification


Product:
 

Mineral
 

Cobalt
 

Copper
 

Iron
 

Manganese
 

Zinc
 

2.8 g.
 

20,000.0 I.U.
 

560.0 g.
 

2.2 g.
 

30.0 g.
 

18.0 g.
 

3.4 g.
 

10.0 g.
 

4000.0 meg.
 

1.7 g.
 

140.0 mg.
 

Source
 

Stabilized vitamin
 

A palmitate
 

Menadione sodium
 

Choline chloride
 

Calcium pantothenate
 

 Per Ton (2,000 Ibs.) of Finished
 

Amount (g)
 

0.4
 

4.0
 

120.0
 

60.0
 

16.0
 

Source
 

Cobalt carbonate
 

Copper sulfate
 

Iron sulfate
 

Manganous oxide
 

Zinc oxide
 

Iodine 1.4 Potassium iodate
 
- 34 ­

These concentrations of vitamins and minerals shall be added to the
 

ration via two separate (vitamin and mineral) premixes with one of the
 

major ingredients as a carrier. The amount of this ingredient used in
 

the final formulation may be adjusted so the total amount of
 

ingredients will equal 100%. Vitamin and mineral concentrations in the
 

premixes shall be made so a minimum of one pound of each premix is
 

required per ton of finished product. In the case of the mineral
 

fortification, the actual amount of each element required is specified.
 Therefore, the amount of each compound used in the premix shall be
 

adjusted according to its mineral concentration.
 

Microanalysis - The total calculated concentration of nutrients in the
 

ration from ingredients and from the fortifications at the time of
 

manufacture shall be as follows:
 

H
 

Crude protein

Crude fat

 % 

% 

Crude fiber % 

Ash % 

Ami no acids
 

(% of total diet) Minimum
 

Arginine 1.25
 

Lysine 1.20
 

Methionine .50
 

Cystine .35
 

Tryptophan .25
 

Glycine 1.10
 

Histidine .50
 

Leucine 1,80
 

Isoleucine 1.10
 

Phenylalanine 1.10
 

Tyrosine .75
 

Threonine .90
 

Valine 1.20
 

Minerals
 

Calcium %
 

Phosphorous %
 

Potassium
 %

%

%
 

Sodi urn
 

Magnesium
 

Minimum 23.5
 

Minimum 5.0
 

Maximum 4.5
 

Maximum 7.0
 

Minimum 1.20
 it
 .95
 i i
 .80
 M
 .33
 

.15
 H
 

Iron PPM
 250.00
 

Zinc PPM n 45.00
 

n
 Manganese PPM 100.00
 n
 Copper PPM 15.00
 M
 Cobalt PPM 0.70
 M
 Iodine PPM 1.80
 
- 35 ­

Vitamins 

Vitamin A 

Vitamin D 

lU/g 

IU/g 

Alpha- tocopherol PPM 

Thiamine PPM 

Minimum it 

H 

n 

15.0 

4.0 

35.0 

14.0 

Riboflavin PPM n 7.0 

Niacin PPM n 80.0 

Pantothenic PPM n 20.0 

acid 

Choline PPM n 2000.0 

Pyridoxine 

Folic acid 

PPM 

PPM 

n 

n 

10.0 

4.0 

Biotin PPM n 0.15 

Vitamin B12 

Vitamin K 

Mcg/lb

PPM 

n 

M 12.0 

3.0 

Approximate Analysis - Analysis for nutrient content of both
 

ingredients and the finished product shall be conducted in accordance
 

with the procedures of the Association of Official Agricultural


Chemists (1965). All nutrient contents shall be expressed as a
 

percentage by weight on an air-dry basis.
 

Ingredient Standards - Ingredients used in the manufacture of this
 

ration will not be contaminated with any more than 3% of foreign
 

materials such as other grains, weed seeds, chaff, etc. Nor will any
 

mold, must, or insect/rodent infestation be allowed. The average
 

minimum nutrient concentrations of ingredients used in the manufacture
 

of this ration shall be equal to the values published in the National
 

Academy of Sciences Publication 1684, United States - Canadian Tables
 

of Feed Composition.
 

Form - The finished product may take the following forms:
 

1.	 Oval shape pellets 5/8"-3/4" wide; 3/8"-7/16" thick; 1"-1 3/8"
 

long
 

2.	 Oval shape pellets 5/8"-3/4" wide; 3/8"-7/16" thick; 3/8" to 1"
 

long
 

3.	 Meal form
 

Special Assays - The product covered by this specification is subject
 

to estrogen assays in accordance with the latest issue of National
 

Institutes of Health Standard No. 2. Estrogen assays shall be
 

initiated by the laboratory once every two weeks. If at any time the
 
- 36 ­

estrogen activity in a batch* of product is found to exceed 4 ppb, such
 

a batch shall be rejected as unsuitable. Periodic analyses of the
 

product also should be made for pesticide, mycotoxin, industrial, and
 

other contaminants. The data from these analyses should be made
 

available to the users of the product.
 

Feed Additives and Processing Restrictions - The product shall contain
 

no antibiotics or estrogen additives of any kind. All milling and
 

warehousing conditions and/or restrictions, as specified in the latest
 

issue of the National Institutes of Health Standard No. 1, apply to
 

the feed covered by this specification. The product shall not be
 

altered in any manner so as to affect the final nutrient content.
 

Marking - Each bag shall be marked (either on the bag or via green tag)
 

with the name of the product, the name of the manufacturer, the net
 

weight, the ingredients, the guaranteed analysis of its contents, the
 

date (month, day, and year) the manufacturing process was completed,
 and the batch number under which it was processed.
 

Packaging - The finished product shall be packaged in commercially
 

acceptable 3-ply laminated paper bags, 50 Ibs. per bag. The paper
 

bags shall be closed by sewing in a manner that will ensure the
 

delivery of uncontaminated animal feed.
 

* For the purpose of this Standard, a "batch" is defined as one
 

continuous production run which may or may not consist of several small
 

batches.
 
- 37 ­

APPENDIX C
 

SAFETY STANDARDS FOR RESEARCH INVOLVING CHEMICAL CARCINOGENS
 

The safety standards provided in this appendix have been adapted from
 

the National Cancer Institute Safety Standards for Research Involving


Chemical Carcinogens (DHEW Publication No. NIH-76-900). These were
 

developed as interim standards for use by personnel employed at the
 

National Cancer Institute (NCI). It is intended that they will be
 

superceded by permanent DHEW safety standards as they become effective.
 The standards contained herein are recommended for use by all 

laboratories involved in research with known, potential, or suspect 

carcinogens. 

I. POLICY
 

The NCI has established a policy that planning and implementation of
 

control practices for the prevention of occupationally-acquired cancer
 

and for the protection of the general environment shall be included in
 

a ll cancer research programs.
 

II. INTRODUCTION
 

The NCI Safety Standards set forth general safety principles that are
 

to be followed in the handling, storage, and disposal of chemical
 

carcinogens. These Standards have been prepared to protect laboratory
 workers and their experiments from inadvertent exposure to chemical
 

carcinogens, as well as to minimize the hazard to the community. They
 include work control practices, environmental control techniques, and a
 

health surveillance program. The Safety Standards are specifically
 designed for work with chemical carcinogens -- which are long-term
 

hazards — and are meant to supplement conventional safety practices,
 such as accident and fire prevention.
 

III. REGULATED CHEMICAL CARCINOGENS
 

Specific chemical carcinogens for which Federal regulation have been
 

promulgated by the Department of Labor are:
 
- 38 ­

A. Low Molecular Weight Aliphatic Derivatives
 

1. Ethylenimine
 

2. beta-Propiolactone
 

3. Vinyl chloride
 

4. Bis (chloromethyl) ether
 

5. Chloromethyl methyl ether
 

B. Aliphatic Nitrosamine Derivatives
 

1. N-Nitrosodimethylamine
 

C. Aromatic Amine Derivatives
 

1. 4-Aminodiphenyl
 

2. 4-Nitrobiphenyl
 

3. Benzidine
 

4. 3,3'-Dichlorobenzidine
 

5. 4,4'-Methylene-bis (2-chloroaniline)
 

6. beta-Naphthylamine
 

7. 2-Acetylaminofluorene
 

D. Aromatic Azo Derivatives
 

1. 4-Dimethylaminoazobenzene
 

IV. CARCINOGEN SAFETY STANDARDS
 

IV.A. Responsibility
 

IV.A.I. Office of Research Safety, NCI
 

The Office of Research Safety shall administer the policy of the
 

NCI for the prevention of occupationally-acquired cancer and for
 

the protection of the general environment. The Office is
 

responsible for: (1) developing standards that are necessary to
 

carry out this policy; (2) assisting program managers and
 

laboratory supervisors in implementing these Standards; (3)
 

coordinating the safety activities of the laboratories of the
 

NCI; (4) developing resources to facilitate the implementation
 

of these Standards; (5) directing the investigation of incidents
 

that result in personnel exposures to chemical carcinogens; (6)
 

maintaining safety records; (7) conducting safety inspections to
 

determine compliance; and (8) ensuring compliance through
 

appropriate administrative channels.
 
- 39 ­

IV.A.2. Laboratory Supervisors
 

Laboratory supervisors are responsible for training their
 

subordinates in safe practices, for correcting work errors and
 

conditions that may result in personal injury, including
 

exposure to chemical carcinogens, and for developing a positive
 

attitude toward safety in laboratory operations on the part of
 

their subordinates. Laboratory supervisors investigate the
 

circumstances surrounding each accident that results in personal
 

injury or exposure to chemical carcinogens; initiate corrective
 

action and recommend to management improvements that ensure
 

maximum safety for their subordinates; and are responsible for
 

preparing safety action plans for the research under their
 

direction. The safety action plans shall describe: (1) the
 

operational practices that will be used to implement these
 

Standards; (2) the emergency procedures to be followed in the
 

event of an accidental exposure to a chemical carcinogen; and
 

(3) the monitoring procedures to assure the effectiveness of the
 

operational practices. The safety action plans shall be
 

approved by the Office of Research Safety. The laboratory
 supervisors shall notify the Office of Research Safety
 

immediately on the occurrence of any accident that results in
 

the exposure of personnel or the environment to a chemical
 

carcinogen.
 

IV.A.3. Laboratory Workers
 

Laboratory workers are responsible for complying with oral and
 

written safety rules, regulations, and procedures required for
 

the performance of the assigned task. This is necessary for the
 

protection of the laboratory workers, other fellow workers, and
 

the public. They also are responsible for reporting to their
 

immediate supervisor all facts pertaining to every accident
 

resulting in personnel injury or exposure to a chemical
 

carcinogen and any action that could result in such incidents.
 

IV.B. Medical Surveillance
 

IV.B.I. Preassignment Examinations
 

An appropriate preassignment physical examination shall be
 

provided each person planning to work with chemical carcinogens.
 The purpose of this examination is to establish a base line
 

against which changes can be measured and to determine whether
 

there exist any medical or other conditions that may lead to an
 

increased risk for the person in the work situation.
 
- 40 ­

IV.B.2. Periodic Examinations
 

A ll employees working with chemical carcinogens shall be
 

provided periodic physical examinations. The purpose of the
 

examinations is to determine whether a change has occurred in
 

the employees' medical state or in other relevant conditions
 

that might lead to increased risk in the work situation. The
 

frequency shall be dependent upon the work circumstances and the
 

general health of the employee.
 

IV.B.3. Records
 

Medical records shall be maintained for the duration of the
 

employee's employment by the NIH Employee Health Service. Upon
 

termination of the employee's employment, including retirement
 

or death, the medical records, or copies thereof, shall be
 

transmitted to the Office of Research Safety which will forward
 

them to the appropriate Office, where they will be maintained
 

for an extended period of time in a manner that will ensure
 

their ready access as needed by the NCI.
 

IV.C. Personnel Practices
 

IV.C.I. Protective Clothing
 

IV.C.I.a. Laboratory Personnel Protective clothing, such
 

as a fully fastened 1aboratory coat, shall be worn in any
 

work area where chemical carcinogens are being used. Gloves
 

that are appropriate to the specific situation shall be used
 

when handling chenical carcinogens. Clean clothing shall be
 

provided daily and shall not be worn outside the work area
 

once the work area has been entered. Clothing contaminated
 

by chemical carcinogens shall be decontaminated or disposed
 

of immediately after an overt exposure. Clothing
 

contaminated with chemical carcinogens shall not be sent out
 

for laundering until decontaminated.
 

IV.C.l.b. Animal Care Personnel Animal care and other
 

personnel entering an area where animals are being treated
 

with chemical carcinogens shall use a complete clothing
 change, including pants and shirts or jumpsuits, shoes or
 

boots, head cover, and gloves. Clean clothing shall be
 

provided daily and shall not be worn outside the work area
 

once it has been entered. Clothing contaminated by chemical
 

carcinogens shall be decontaminated or disposed of
 

immediately after an overt exposure.
 
- 41 ­

IV.C.2. Protective Equipment
 

Personnel engaged in animal or other procedures where exposure
 

to airborne particulates contaminated with chemical carcinogens
 

could occur shall wear an appropriate face mask or respirator.
 The selection of an appropriate face mask or respirator shall be
 

made according to the guidance of the Office of Research Safety.
 The face mask or respirator shall not be worn outside the work
 

area. Used filters shall be disposed of and the respirator
 

housing shall be decontaminated daily.
 

IV.C.3. Showers
 

Personnel engaged in animal procedures or any other procedure
 

where exposure to airborne particulates contaminated with
 

chemical carcinogens could occur shall shower after each exit
 

from the work area. All personnel shall shower immediately
 

after any overt exposure to a chemical carcinogen.
 

IV.C.4. Eating, Drinking, and Smoking
 

There shall be no eating, drinking, smoking, chewing of gum or
 

tobacco, application of cosmetics, or storage of food in areas
 

where chemical carcinogens are used.
 

IV.C.5. Pipetting
 

Mechanical pipetting aids shall be used for all pipetting
 

procedures. Oral pipetting shall be prohibited.
 

IV.C.6. Personal Hygiene
 

A ll personnel shall wash their hands immediately after
 

completion of any procedures in which chemical carcinogens have
 

been used.
 

IV.D. Operational Practices
 

IV.D.I. Work Area Identification
 

Entrances to all work and storage areas where chemical
 

carcinogens are present shall be posted with signs bearing the
 

legend:
 

DANGER—CHEMICAL CARCINOGEN
 

Authorized Personnel Only
 
- 42 ­

IV.D.2. Access Control
 

Work and storage areas where chemical carcinogens are present
 

shall be entered only by personnel authorized by the Laboratory
 

Supervisor. Access procedures shall be prominently displayed at
 

points of access.
 

IV.D.3. Work Surfaces
 

A ll work surfaces (bench tops, hood floors, etc.) on which
 

chemical carcinogens are used shall be covered with stainless
 

steel or plastic trays, uncracked glass plates, dry absorbent
 

plastic-backed paper, or other impervious material. The
 

protective surfaces shall be examined for possible contamination
 

immediately after the procedure involving the chemical
 

carcinogen has been completed. The contaminated surface shall
 

be decontaminated or disposed of as is appropriate.
 

IV.D.4. Use of Laboratory-Type Hoods
 

Procedures that involve the use of chemical carcinogens shall be
 

conducted in a laboratory-type hood or other suitable
 

containment device when: (1) the procedure involves the use of
 

volatile chemical carcinogens; or (2) the procedure results in
 

the generation of aerosols, such as from the opening of closed
 

vessels, transfer operations, weighing, preparation of feed
 

mixtures, and the application, injection and intubation of a
 

chemical carcinogen to experimental animals. Each
 

laboratory-type hood or containment device used for containment
 

of chemical carcinogens shall display a label bearing the
 

legend:
 

DANGER—CHEMICAL CARCINOGEN
 

IV.D.5. Working Quantities
 

Only minimum working quantities of chemical carcinogens shall be
 

present in a work area.
 

IV.D.6. Identification, Storage, and Inventory
 

IV.D.6.3, Labeling Storage vessels containing chemical
 

carcinogens shall be labeled:
 

DANGER—CHEMICAL CARCINOGEN
 
- 43 ­

IV.D.S.b. Storage Stock quantities of chemical
 

carcinogensshall be catalogued and stored in a specific
 

storage area that is secured at all times.
 

IV.D.6.C. Inventory An inventory of all chemical
 

carcinogens shall be maintained by the Laboratory
 Supervisor. The inventory records shall include the
 

quantities of chemical carcinogens acquired, dates of
 

acquisition, and disposition. A copy of the inventory
 

records shall be furnished to the Office of Research Safety
 

on a semi-annual basis.
 

IV.D.7. Laboratory Transport
 

IV.D.7.a. Stock Quantities of Chemical Carcinogens An
 

unbreakableoutercontainershallBeusedt o transport
 

chemical carcinogens.
 

IV.D.7.b. Contamina ted Materia1s Materials contaminated
 

with chenriclTlcarcinogensthat are transferred from work
 

areas to disposal areas shall be placed into separate
 

closed, plastic bags, or other suitable impermeable, and
 

sealed containers for each carcinogen and labeled with both
 

the name of the carcinogen and "DANGER—CHEMICAL
 

CARCINOGEN," before being transported.
 

IV.D.8. Houskeeping
 

General housekeeping procedures which suppress the formation of
 

aerosols, such as the use of a wet mop or a vacuum cleaner
 

equipped with a HEPA filter on the exhaust, shall be used. Dry
 

sweeping and dry mopping are prohibited because of the hazard of
 

aerosol formation. In those instances where a 

chemical-containing material is spilled, special procedures 

shall be followed. 

IV.D.9. Protection of Vacuum Lines
 

Each vacuum service shall be protected with a disposable HEPA
 

filter and liquid trap to prevent entry of any chemical
 

carcinogen into the vacuum system. When using a volatile
 

carcinogen a separate vacuum pump or other device shall be used
 

in conjunction with an appropirate laboratory-type hood or other
 

containment device approved by the Office of Research Safety.
 
- 44 ­

IV.D.10. Packaging and Shipping
 

The packaging and shipping methods established by the Department
 

of Health, Education and Welfare for the transportation of
 

etiologic agents (42 CFR 72.25, 1972) shall be applied to the
 

shipment of all stable chemical carcinogens. For chemical
 

carcinogens which are physically or chemically unstable, e.g.,
 corrosive, explosive, or flammable, the procedures established
 

by the Department of Transportation for the handling of such
 

materials (49 CFR 173, 1973) shall be employed.
 

IV.D.ll. Decontamination and Disposal
 

IV.D.11.a. Decontamination Contaminated materials shall
 

be decontaminated by procedures that either inactivate the
 

chemical carcinogens or remove them for subsequent disposal.
 

IV.D.ll.b. Disposal Contaminated wastes, cleaning
 

devices, and animal carcasses shall be collected in
 

impermeable containers, which are closed prior to removal
 

from the work areas, and disposed of by appropriate methods
 

as approved by the Office of Research Safety.
 

IV.D.ll.c. Spills Chemical carcinogens which have spilled
 

out of a primary container so as to constitute a hazard
 

shall be activated -In situ or shall be absorbed by
 

appropriate means for subsequent disposal.
 

IV.D.12. Animal Housing
 

Animals shall be housed in a cage that confines feed, feces,
 urine, and bedding within the enclosure. For non-volatile
 

carcinogens, use of a cage with solid sides and bottom in
 

conjunction with a filter top is recommended. When using a
 

volatile chemical carcinogen the cage must be used in
 

conjunction with appropriate ventilation systems. Alternative
 

animal housing methods shall require approval by the Office of
 

Research Safety.
 
- 45 ­

IV.E. Laboratory-Type Hoods and Exhaust Air-Treatment
 

IV.E.I. Laboratory-Type Hoods
 

IV.E.I.a. Open Face Hoods Open face hoods, such as a
 

chemical fumi hood, slha 11 have an average linear face
 

velocity of 100 feet per minute. The minimum air velocity
 

at any point in the face of the fully opened hood shall not
 

be less than 85 feet per minute.
 

IV.E.l.b. Glove Boxes Glove boxes shall be kept under
 

negative air pressure of 0.5 inches water gauge with respect
 

to the space in which they are located.
 

IV.E.I.e. Laminar Flow Biological Safety Cabinet Laminar
 

flow biological safety cabinets may be used for the
 

containment of in vitro procedures involving the use of
 

chemical carcinogens provided that: (1) the exhaust air
 

flow is sufficient to provide an inward air flow at the face
 

opening of the cabinet equal to 100 feet per minute times
 

the face opening area, (2) contaminated air plenums that are
 

under positive air pressure are leak tight, and (3) the
 

cabinet exhaust air is discharged outdoors.
 

IV.E.2. Exhaust Air Treatment
 

The exhaust air from laboratory type hoods and other ventilated
 

containment devices shall be appropriately treated such as by
 

filtration, reaction, absorption, adsorption, incineration, or
 

dilution, so that the concentration of any chemical carcinogen
 

or combination of chemical carcinogens in the final effluent
 

which is discharged outdoors shall not exceed 1 ppb or natural
 

background levels, whichever is greater. Exhaust air treatment
 

systems that remove chemical carcinogens from the exhaust air by
 collection mechanisms such as filtration, absorption, and
 

adsorption shall be operated in a manner that permits
 

maintenance so as to avoid direct contact with the collection
 

medium.
 

IV.E.3. Performance Certification
 

Performance of laboratory-type hoods and exhaust air treatment
 

systems shall be certified at least annually.
 
- 46 ­

IV.F. Facilities
 

IV.F.I. General Ventilation Control
 

IV.F.I.a. General Exhaust Air The general exhaust air
 

from work areas in1 which chemical carcinogens are used shall
 

be discharged outdoors and dispersed to the atmosphere so as
 

to prevent re-entry into the facility. No recirculation of
 

exhaust air from work areas is permitted.
 

IV.F.l.b. Air Pressure Work areas in which chemical
 

carcinogens are used shall be kept under negative air
 

pressure with respect to the access corridor. For
 

facilities where work areas have "clean" access corridors
 

and "dirty" egress corridors, the "dirty" egress corridor
 

shall be kept under negative air pressure with respect to
 

the work area.
 

IV.F.2. Change Rooms and Showers
 

Clothing change rooms and showers shall be available to
 

personnel engaged in activities involving chemical carcinogens.
 

V. GLOSSARY
 

Given below are some definitions of terms used in the NCI
 

Safety Standards for Research Involving Chemical
 

Carcinogens.
 

ACCIDENT -- is an unforseen circumstance or set of
 

circumstances resulting in the release of a chemical
 

carcinogen that may result in contact with or exposure
 

to a chemical carcinogen.
 

AUTHORIZED PERSONNEL -- are those employees whose duties
 

require them to be in work or storage areas where
 

chemical carcinogens are present, and who are
 

specifically assigned there by the Laboratory Supervisor.
 

CHEMICAL CARCINOGEN — is a chemical that has been
 

demonstrated to cause tumors in mammalian species by
 

induction of a tumor type not usually observed; or
 
- 47 ­

by induction of an increased incidence of a tumor type
 

normally seen, or by its appearance at a time earlier
 

than would be otherwise expected.
 

DECONTAMINATION — is the safe removal of a chemical
 

carcinogen from a contaminated item.
 

DISPOSAL — is the safe elimination of a chemical
 

carcinogen from the general environment by inactivation,
 degradation, destruction, or other appropriate method.
 

EMERGENCY -- is an accident or other event that requires
 

immediate action.
 

GLOVES — are covers to protect the hands of a worker
 

against contact with or exposure to a chemical
 

carcinogen.
 

GLOVE BOX — is a fully enclosed ventilated containment
 

device, provided with port-holes to which are
 

attached flexible gloves that permit the insertion of
 

hands within the device, to perform manual operations
 

without contact with or exposure to a chemical
 

carcinogen.
 

HEPA FILTER -- is a High Efficiency Particulate Air
 

Filter that is capable of retaining 99.97% of a mono
 

dispersed aerosol of 0.3 m particles.
 

LABORATORY SUPERVISOR -- is a person who plans and
 

directs research projects and other activities in the
 

laboratory.
 

LABORATORY WORKER — is a person who carries out an
 

assigned task in the laboratory.
 

LAMINAR FLOW BIOLOGICAL SAFETY CABINET — is a ventilated
 

containment device that provides both personnel
 

protection and a contamination-free work environment.
 Personnel protection is achieved by an inflow of room air
 

at the work opening where it is quickly entrained in a
 

recirculating air stream and removed through an exhaust
 
- 48 ­

grille at the leading edge of the work area. A
 

contamination-free work zone is achieved by supplying air
 

through High Efficiency Particulate Air Filters downward
 

towards the work surface at a uniform velocity. Air flow
 

equal to the in flow of room air is exhausted through
 

High Efficiency Particulate Air Filters incorporated in
 

the cabinet or the exhaust system.
 

MONITORING -- is to determine, on a regular basis,
 the possible presence of a specific chemical
 

carcinogen in the environment by means of a
 

sensitive analytical method.
 

OPEN FACE LABORATORY TYPE HOOD -- is a device enclosed on
 

three sides and the top and bottom; is designed and
 

maintained to draw air inward at an average linear face
 

velocity of 100 feet per minute; and is designed,
 constructed, and maintained so that an operation
 

involving a chemical carcinogen within the hood does not
 

require the insertion of any portion of a worker's body
 

other than his hands and arms.
 

PROTECTIVE CLOTHING — are clothes designed to protect a
 

worker against contact with or exposure to a chemical
 

carcinogen.
 

PROTECTIVE EQUIPMENT — is equipment in addition to
 

protective clothing and gloves, such as a face mask or a
 

respirator, that is designed to protect a worker against
 

contact with or exposure to a chemical carcinogen.
 

SAFETY ACTION PLAN — is a plan that describes the
 

procedures and mechanisms, including operational
 

practices, emergency procedures, and monitoring
 

techniques, to be used in implementing the NCI Safety
 

Standards for Research involving Chemical Carcinogens.
 

STORAGE AREA — is the area where a chemical carcinogen
 

not required for daily usage is stored.
 

WORK AREA — is an area where entry and exit is
 

restricted and controlled because of the presence of a
 

chemical carcinogen.
 
- 49 ­

WORKING QUANTITY — is that amount of chemical carcinogen
 

required for carrying out a project on a day-to-day
 

basis.
 
- 50 ­

APPENDIX D
 

ANIMAL RANDOMIZATION
 

Randomization of the animals is necessary to ensure that unintentional
 

selection biases are not introduced into the bioassay study. All
 

the animals should be from the same supply source and, ideally, within
 

2-3 days of the same age. Unsuitable animals should be culled
 

and discarded during the quarantine period (see Section III.C.).
 Optimally, animals should be assigned to experimental groups and
 

cages by the use of a table of random numbers. In this instance,
 each animal must be consecutively numbered. An example of
 

such a randomization procedure is shown below.
 

Random assignment is to be made of 150 animals to three experimental
 

groups so that there are 50 in each group. The animals are
 

consecutively numbered. A starting place in a table of random
 

numbers is selected and successive triplets of numbers listed, omitting
 

000, numbers greater than 150, and repeats. The first 50 numbers
 

correspond to animals assigned to group one, the second 50 to group
 

two, and the remaining 50 to the third group. When animals are
 

segregated into weight distribution groups prior to randomization,
 this procedure is repeated completely for each of these groups.
 This will ensure that there will be an equal number of animals from
 

each weight distribution group in each of the experimental groups.
 
- 51 ­

APPENDIX E
 

BIOASSAY PATHOLOGY PROCEDURES
 

These Bioassay Pathology Procedures have been prepared by Dr. Robert
 

A. Squire, Head, Tumor Pathology Section, Carcinogenesis Program,
 Division of Cancer Cause and Prevention, National Cancer Institute.
 

The purpose of this document is to assure uniform and scientifically
 

acceptable pathologic procedures in the NCI Carcinogenesis Bioassay
 

Program. It is to be implemented in all studies in which the effects
 

of possible carcinogens may be systemic, regardless of the route of
 

administration. The procedures are not necessarily intended for
 

specific target-tissue Carcinogenesis studies or for other toxicologic
 

evaluations.
 

I. Personnel
 

A.	 A board-certified veterinary or medical pathologist with
 

experience in laboratory animal pathology shall be responsible
 

for all pathology procedures, evaluations, and reporting. If
 

not board-certified, persons may be acceptable if appropriate
 

training and experience judged to be satisfactory by NCI can be
 

demonstrated.
 

B.	 Histology technician(s) shall be supervised by an HT/ASCP
 

registered technician, judged to be qualified by NCI. If not
 

certified, persons may be acceptable if appropriate training
 

and experience judged to be satisfactory by NCI can be
 

demonstrated.
 

C.	 Prosectors shall be trained and experienced in laboratory
 

animal dissection and able to recognize and describe gross
 

abnormalities. Careful performance of the necropsy for the
 

detection of possible tumors at any site is vital to
 

Carcinogenesis experiments. Therefore, qualified and
 

well-supervised personnel are necessary.
 

D.	 Personnel must be available for weekend coverage to necropsy
 

dead or moribund animals.
 
- 52 ­

II. Facilities
 

A.	 Refrigeration shall be available for holding dead animals until
 

necropsy. Animals should not be frozen.
 

B.	 The gross necropsy facility should be in close proximity to the
 

pathologist's office(s) and equipped with adequate working
 

surfaces, dissection boards, running water with drains,
 adequate lighting, ventilation, exhaust hoods and gross
 

photography capabilities.
 

C.	 The histology laboratory shall be separate from the necropsy
 

area and equipped with automatic tissue processor, microtomes,
 embedding and staining equipment, and supplies.
 

D.	 Adequate storage facilities are required to store and file
 

histologic slides, tissue blocks, and wet tissues for duration
 

of the contract. This facility should be vermin proof and
 

temperature controlled.
 

E.	 An area for trimming of fixed tissues, with adequate
 

ventilation and exhaust hoods, is required.
 

III. Gross Necropsy
 

A.	 All animals which die or are sacrificed in the repeated dose,
 subchronic, and chronic studies shall be necropsied (unless
 

cannabilism or autolysis precludes the examination).
 

B.	 The gross dissection and evaluation shall be performed by or
 

under the direct supervision of the pathologist(s).
 

C.	 Th necrops e y is defined as external examination, including body
 

orifices, and examination and fixation of all of the following
 

tissues:
 

Gross lesions


Tissue masses or suspect tumors


& regional lymph nodes


Skin


Mandibular lymph node


Mammary gland


Salivary gland


Larynx


Trachea


 Lungs & bronchi
 

 Heart
 

 Thyroids
 

 Parathyroids
 

 Esophagus
 

 Stomach
 

 Duodenum
 

 Jejunum
 

 Ileum
 
- 53 ­

Cecum Spleen
 

Colon Kidneys
 

Rectum Adrenals
 

Mesenteric lymph node Bladder
 

Liver Seminal vesicles
 

Thigh muscle Prostate
 

Sciatic nerve Testes
 

Sternebrae, vertebrae, Ovaries
 

or femur (plus marrow) Uterus
 

Costochondral junction, Nasal cavity
 

rib Brain
 

Thymus Pituitary
 

Gallbladder Eyes
 

Pancreas Spinal cord
 

D.	 Specific Methods
 

1.	 Peripheral blood smears shall be prepared from all animals
 

at sacrifice, immediately air-dried, and fixed in absolute
 

methanol within 24 hours.
 

2.	 All tissues and/or organs are to be examined in situ, then
 

dissected from the carcass, reexamined, including cut
 

surfaces, and fixed in 10% neutral buffered formalin.
 

3.	 Lungs of mice and rats may be fixed in their entirety after
 

opening and examining the trachea and mainstem bronchi.
 

4.	 Th calvariu e m shall be removed and the dorsal nasal bone
 

removed for examination of nasal turbinates. The entire
 

skull may be fixed with the brain in situ.
 

5.	 Other tissues shall be fixed at a thickness not exceeding
 

0.5 cm.
 

6.	 Distended urinary bladders shall be opened and examined
 

before fixation. Contracted, empty bladders shall be
 

partially distended with formalin and opened and examined
 

after fixation.
 

7.	 Liver lobes shall be sliced, the kidneys longitudinally
 

bissected, and the cut surfaces examined before fixation.
 

8.	 The entire mucosal surfaces of the esophagus, stomach,
 small and large intestines, and rectum shall be opened and
 

examined before fixation.
 
- 54 ­

9.	 Multiple, representative portions of large or variable
 

tissue masses including surrounding unaffected tissues
 

shall be fixed.
 

10.	 Several thoraco-lumbar vertebrae shall be fixed with the
 

spinal cord in situ.
 

E.	 Al gros l s lesions shall be recorded in narrative, descriptive
 

terms including location, size (in mm), number, shape, color,
 and	 texture.
 

F.	 Carcasses of animals may be discarded immediately following
 

necropsy and fixation of all tissues listed under III C.
 

IV. Fixed Tissue Trimming
 

A.	 It is desirable that fixation time be no less than 48 hours nor
 

more than 12 weeks.
 

B.	 Tissue trimming shall be performed by or under the supervision
 

of the pathologist(s) with the gross necropsy descriptions
 

available. Additional gross observations shall be recorded at
 

the time of trimming if necessary.
 

C.	 Tissues shall be trimmed to a maximum thickness of 0.3 cm for
 

processing.
 

D.	 Specific methods
 

1.	 Multiple portions of tumors or masses shall be submitted if
 

these are large or variable in appearance. Surrounding normal
 

tissue shall be included.
 

2. Parenchyma! organs, e.g., liver, shall be trimmed to allow
 

the largest surface area possible for examination.
 

3. Mid-longitudinal sections through the entire cortex and
 

medulla of each kidney shall be submitted.
 

4. Entire coronal (a transverse section parallel to the long
 

axis of the body) sections of both right and left lungs
 

including main-stem bronchi shall be submitted.
 

5. Three cross sections of brain shall include: (a) frontal
 

cortex and basal ganglia, (b) parietal cortex and thalamus, and
 

(c)	 cerebellum and pons.
 

6. Hollow organs shall be trimmed and blocked to allow a
 

cross-section slide from mucosa to serosa.
 
- 55 ­

V. Histologic Technique 

A.	 Tissues shall be cut at 4-6 y and stained with hematoxylin and
 

eosin (H & E).
 

B.	 Blood smears shall be air-dried at necropsy, then fixed in
 

absolute methanol for five minutes. Smears from animals with
 

anemia, enlarged thymus, lymphadenopathy, or hepatosplenomegaly
 

shall be stained with Wrights, Giemsa, or similar Romanovsky
 

stain.
 

VI. Histopathologic Examinations
 

A.	 Animals to be subjected to histopathologic examination:
 

1. All control animals (see * on page 56), highest dose-level
 

animals without mortality, and the next higher dose-level group
 

in subchronic studies.
 

2.	 All control (see * on page 56) and treated animals in
 

chronic studies.
 

B. Histopathologic examination is defined as histologic examina­ tion of the following:
 

gross lesions	 1 i ver
 

tissue masses or suspect tumors & gallbladder
 

regional lymph node(s) pancreas


blood smear (if anemia, enlarged thymus, spleen
 

lymphadenopathy or hepatosplenomegaly, kidneys


is present) adrenals
 

mandibular lymph node bladder
 

mammary gland prostate
 

salivary gland testes
 

sternebrae, femur or vertebrae ovaries
 

including marrow uterus
 

thymus	 brain (three sections
 

trachea including frontal cortex
 

lungs & mainstem bronchi & basal ganglia, parietal
 

heart cortex & thalamus, &
 

thyroids cerebellum & pons)
 

parathyroids pituitary
 

esophagus eyes (if grossly abnormal)
 

stomach spinal cord (if neurologic
 

small intestine (one section) signs are present)
 

colon
 
- 56 ­

VII. Pathology Reports (see Individual Animal Data Record, p. 28)
 

A.	 Descriptive narratives of gross necropsy findings are required
 

for all animals. The number as well as description of tissue
 

masses should be included. If these are confluent or too
 

numerous to count (TNTC), this should be indicated.
 

B.	 One narrative histologic description per animal test group of
 

each different histopathologic diagnosis is required. This may
 

be done the first time a given lesion (including tumors of the
 

same type) is observed in a group.
 

C.	 Histopathologic diagnoses of all lesions shall be entered under
 

Organ and Diagnosis. Indicate primary versus metastatic
 

tumors, e.g.:
 

1.	 Liver, hepatocellular carcinoma
 

2.	 Lung, hepatocellular carcinoma, metastatic
 

3.	 Kidney, interstitial nephritis, chronic
 

VIII. To be Retained until Termination of Study and Final Report
 

A.	 All wet tissues, preferably plastic-bagged, shall be clearly
 

and permanently labeled and retained in a vermin-proof area.
 

B.	 Al histologi l c slides and paraffin blocks shall be retained
 

under suitable temperatures.
 

C.	 N materia o l shall be discarded without prior permission of NCI.
 

*	 Positive control animals shall be subject to complete
 

gross necropsy and tissues fixed as in III C. Tissues to
 

be subjected to histopathologic examination will be
 

determined by consultation with the NCI project officer
 

and will be sufficient to determine tumor types and
 

incidence.
 
- 57 ­

IX. Pathology Material to be Submitted to the NCI
 

A.	 One H & E stained slide plus the tissue block representative of
 

each different neoplasm or treatment-related lesion from each
 

chemical test group shall be sent to the Tumor Pathology
 

Section when the test is completed or sooner, if possible, or
 

if consultation is desired.
 

B.	 All pathologic specimens are the property of NCI and shall be
 

submitted to NCI upon request.
 
- 58 ­

APPENDIX F
 

CARCINOGEN BIOASSAY INFORMATION
 

For each bioassay study done on a particular chemical, the information
 

listed below should be collected. This information conforms with the
 

recommendations made in the report by the Panel on Carcinogenicity


Testing of the International Union Against Cancer (Berenblum, 1969).
 

GENERAL: 1. Outline of the bioassay study
 

2.	 Bioassay study number in the investigator's
 

file
 

3.	 Names of the investigators responsible for
 

the bioassay study, including histopatho­ logical diagnoses
 

4.	 Name of bioassay laboratory
 

CHEMICAL: 1. Name, Chemical abstract number, NCI number
 

2.	 Name, synonyms
 

3.	 Formula
 

4.	 Source (generic)
 

5.	 Manufacturer
 

6.	 Batch number
 

7.	 Date(s) when received
 

8.	 Storage (before its reception)
 

9.	 Physical state and other characteristics
 

10. Melting point
 
- 59 ­

11. Solubility
 

12. Criteria of purity
 

13. Impurities (generic)
 

14. Methods of synthesis
 

15. Storage conditions and dates
 

16. Other
 

PREPARATION: 1. Chemical(s): name, data sheet number
 

2. Vehicle(s): name, data sheet number
 

3. Preparation and concentrations
 

4. Methods of preparation
 

5. Amount prepared each time
 

6. Frequency of preparation
 

7. Physical state
 

8. pH
 

9. Stability and decomposition
 

10. Storage
 

11. Date(s) prepared
 

12. Other
 

ANIMALS: 1. Species
 

2. Strain and subline
 

3. Initial number by sex (male and female)
 

4. Date(s) of birth (male and female)
 

5. Source
 

a. Own colony (give reference)
 

b. Other
 
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6.	 Breeding
 

a.	 Inbred
 

b.	 Random
 

c.	 Outbred
 

d.	 Other
 

7.	 Disease Control
 

a. Specific pathogen-free 

b. Germ-free 

c.	 Conventional
 

d.	 Vaccinated
 

8.	 Distribution in groups
 

a.	 Pooled at weaning
 

b.	 Random
 

c.	 Random tables
 

d.	 Littermates
 

e.	 Other
 

9.	 Other experimental groups included in the
 

same distribution
 

10. Initial number per cage
 

11. Divided by sex
 

12. Age when obtained from Animal House
 

13. Maintenance (general conditions)
 

a.	 Ow standar n d (give reference)
 

b.	 Special
 

14. Cages
 

15. Bedding
 

16. Room temperature (range)
 

17. Light
 

a.	 Source
 

b.	 Time cycle
 

18. Diet
 

a.	 Type
 

b.	 Source
 

19. Amount of diet
 

a.	 Ad libitum
 

b.	 Measured
 
- 61 ­

20. Water
 

a. Tap
 

b. Other
 

21. Amount of water
 

a. Ad libitum
 

b. Measured
 

22. Other or special conditions
 

TREATMENT: 1. Special pretreatment conditions
 

2. Treatment multiplicity
 

a. Single type
 

b. Combined
 

Item 3-1 4 should apply for each treatment
 

3. Preparation administered
 

4. Dose per administration
 

a. Volume
 

b. Weight
 

c. How measured
 

5. Route
 

6. Site
 

7. Methods and instruments used
 

a. Surgical procedures
 

b. Anesthesia
 

c. Sterility
 

8. Frequency of administration
 

9. Total number of doses
 

10. Total dose given
 

11. Total time of treatment
 

12. Date treatment started
 

13. Date treatment ended
 

14. Others
 
- 62 ­

PLAN OF
 

OBSERVATIONS: 1. Age of animals at start of experiment
 

2. Weight of animals at start of experiment
 

3. Duration of experiment
 

a. Lifespan
 

b. Interruptions (from when to when and why)
 

4. Frequency of checking
 

5. Frequency of weighing
 

6. Frequency of charting
 

7. Frequency of measuring consumption of:
 

a. Feed
 

b. Water
 

8. Other observations
 

9. Autopsies
 

a. On all animals
 

b. With the exception of: 

(1) decomposed animals
 

(2) lost animals
 

(3) other
 

10. Autopsy examinations
 

a. Complete
 

b. Except cranial cavity
 

c. Other exceptions
 

11. Histology
 

a. All tumors (note exceptions)
 

b. Other tissues
 

12. Other pathological observations
 

CONTROLS: 1. List:
 

a. Each group
 

b. Selection
 

VARIATIONS: 1. List protocol additions or changes
 
- 63 ­

REPORTS: 1. Animal groups 

a. Body weight curves 

2. Individual animals 

a. Identification number 

b. Mode of death (died or sacrificed) 

c. Time of death (in days or weeks of age, or time from start of bioassay study) 

d. Diagnosis of tumors found at necropsy 

and other pertinent pathology 

e. Indication if necropsy not done (as in 

decomposition) and animal is considered 

lost from the study 
- 64 ­

GENERAL REFERENCES
 

Balazs, T., Measurement of Acute Toxicity, In Methods in
 

Toxicology, ed. G. E. Paget. F. A. Davis Co.,
 Philadelphia, Pa., 1970, pp. 49-81.
 

Benitz, K. R., Measurement of Chronic Toxicity, In
 

Methods in Toxicology, ed. G. E. Paget, F. A. Davis
 

Co., Philadelphia, Pa., 1970, pp. 82-131.
 

Berenblum, I. (ED.): Carcinogenicity Testing. UICC
 

Technical Report Series. Switzerland, Imprimerie G. de
 

Buren S. A., Geneve, 1969, Volume 2, p. 56.
 

Committee on Nomenclature and Classification of Disease:
 

Systematized Nomenclature of Pathology , Chicago, College
 

of American Pathologists, 1965, p. 439.
 

Fitzhugh, 0., Garth, Subacute Toxicity, In Appraisal of
 

the Safety of Chemicals in Foods3 Drugs and Cosmetics,
 

Edited and published by The Editorial Committee of the
 

Assoc. of Food & Drug Officials of the U.S., 2411 N.
 Charles St., Baltimore, Md., 1959, pp. 26-35.
 

Food and Drug Administration Advisory Committee on
 

Protocols for Safety Evaluation: Panel on Carcinogenesis
 

Report on Cancer Testing in the Safety Evaluation of Food
 

Additives and Pesticides, Toxicol. Appl. Pharmacol.
 

20, 419-438, 1971.
 

Hagen, E. C., Acute Toxicity, In Appraisal of the Safety
 

of Chemicals in Foods, Drugs and Cosmetics, Edited and
 

published by The Editorial Committee of the Assoc. of
 

Food & Drug Officials of the U.S., 2411 N. Charles St.,
 Baltimore, Md., 1959, pp. 17-25.
 

Linhart, M. S., Cooper J. A., Martin, R. L., Page, N.
 P. and Peters, J. A., Carcinogenesis Bioassay Data
 

System. J. Comp. Biomed. Ees. 7, 230-248, 1974.
 

Litchfield, J. T. and Wilcoxon, F. A., A simplified
 

method of evaluating dose-effect experiments, J.
 Pharmacol. Exp. Ther., 96, 99-113, 1949.
 

Magee, P. N., Tests for Carcinogenic Potential, In
 

Methods in Toxicology, ed. G. E. Paget, F. A. Davis
 

Co., Philadelphia, Pa., 1970, pp. 158-196.
 
- 65 -


Subcommmittee on Carcinogenesis, Food Protection
 

Committee, Food and Nutrition Board, National Academy of
 

Sciences - National Research Council; Problems in the
 

Evaluation of Carcinogenic Hazards from Use of Food
 

Additives, Cancer Res. 21, 429-456, 1961.
 

Weisburger, John H. and Weisburger, Elizabeth K., Tests
 

for Chemical Carcinogens, In Methods in Cancer Research,
 

vol. I, ed. Harris Busch. Academic Press, New York,
 1967, pp. 307-398.
 

^U.S.GOVERNMEN T PRINTIN G OFFICE : 1976--217-2^ 2 

DHEW Publication No. (NIH) 76-801