November 1974
INDUSTRY SURVEY OF TEST METHODS
OF POTENTIAL HEALTH HAZARD
By
Geoffrey Woodard, Ph.D.,
Woodard Research Corporation
12310 Pinecrest Road
Herndon, Virginia 22070
Contract No. 68-01-2104
Project Officer
Elton R. Homan
Office of Toxic 'Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
Prepared for
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
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This report has been reviewed by the Office of Toxic
Substances, EPA, and approved for publication. Approval
does hot signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency,
nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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The following article, "Toxicology: Cost/Time", by Gehring et al,
is reprinted from Fd. Cosmet. Toxicol. 11: 1097-1110 (19737~by
permission of Pergamon Press.
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CONTENTS
Page
Abstract i
Acknowledgements iii
Sections
I Conclusions 1
II Recommendations 3
III Introduction 5
- Definition of the Problem
- Need for the Study
- Study Purpose
/
IV Methods Employed 8
A. Sample Selection Process
- Selection of Organizations to
be Considered
o Standard Industrial Classifi-
cation Codes
o Selection of Potential Organi-
zations
o Cross Checks from Professional
Society Registers
o Influence of Trade Associations
- Initial Contact Procedure
- Follow-up Requests
- Selection of Final Participants
B. Visit to Site and Interview
- Preparation of Outline for
Interview
- Conduct of Interview
- Collection of Available Documents
C. Ordering and Summarizing Data for
Each Company
D. Use of Ancillary Information
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V. Evaluation of the Present Industry 16
Practices
A. Decision Making Related to
Toxicological and Environmental
Evaluations
- New Products
- Existing Products
- Product Changes
o New Methods of Production
o Use of Alternate Sources of
Raw Materials
B. Development and Use of Specific
Experimental Methodology
- Toxicological Methods
- Environmental Methods
C. Monitoring Systems
— Consumer Complaints
- Marketing Liaison on New Appli-
cations
- When to Expand Toxicological
and Environmental Testing
D. Chemical Purity
E. Costs for Conducting Toxicological
and Environmental Hazards Assessment
VI Identification of Problem Areas Existing
in Toxic Substances Evaluation 4^
A. New Products
B. Existing Products
VII References 44
VIII Appendices 46
A. Industrial Profits of Companies
Included in Survey
B. Toxicology: Cost/Time
C. Protocols
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ABSTRACT
Many new and existing industrial chemicals enjoy uses that
are either not regulated or are inadequately regulated under
State or Federal law in order to prevent possible damage
to man and his environment. The Toxic Substances Control
Act concerns itself with this area of chemical use. Regu-
lations to implement this act when it becomes law will be
promulgated. In order to design appropriate regulations,
it is necessary to'have a knowledge of the state-of-the-art
as practiced by the chemical industry at the present time .
for evaluation of potential adverse health and environmental
effects of new and existing chemicals.
Nine companies selected as representing a cross section of
prominent producers of chemicals identified through four-
digit Standard Industrial Classification (SIC) codes were
surveyed with respect to methods used to assess toxico-
logical and environmental properties of new and existing
chemicals.
Four progressive levels of investigational effort depending
upon extent, frequency, and nature of chemical use are
identified:
o Single or infrequent exposure - Acute and irritation
tests
o' Occasional low-level exposure - Short-term repeated
dose, sensitization, fish and bird studies
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o Frequent low-level, occasional high-level exposures -
Teratology, mutagenicity, metabolism, biodegradation,
environmental transport studies
o Frequent high-level, general consumer, unavoidable
exposures - Reproduction, lifetime, carcinogenic,
environmental fate, food chain studies
Factors influencing the decisions regarding the level(s) of
investigation needed or the initiation of the next higher
level series of studies are explored. No mathematical for-
mula has emerged to specify the level of testing required.
A chemical-by-chemical assessment by competent pharmacol-
ogists, toxicologists, and chemists is still needed.
Lack of a mechanism to trigger expanded levels of testing
is a serious problem.
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This report was submitted in fulfillment of Contract No.
68-01-2104 by Woodard Research Corporation under the
sponsorship of the Environmental Protection Agency. Work
was completed as of May 15, 1974.
ACKNOWLEDGMENTS
Initial company contacts, selection of SIC codes, and
computer sorts were conducted primarily by Applied Manage-
ment Sciences (AMS), 962 Wayne Avenue, Silver Spring,
Maryland 20901, subcontractors to Woodard Research Corpora-
tion. Mr. Locke Bogart and/or Mr. Herbert Cantor of AMS
also participated in the majority of the site visits.
XII
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I. CONCLUSIONS
1. The chemical industry has developed policies and op-
erating procedures that assure no new chemical will reach
the marketplace without some degree Of testing for poten-
tial toxicological and environmental hazard.
2. As the use of a chemical already existing increases,
there is essentially no mechanism that triggers expanded
toxicological and environmental testing. Such expanded
testing when it occurs is nearly always in response to a
reaction to some adverse finding outside the company.
3. Four progressive levels of investigations of new chem-
icals have been identified that depend upon the extent,
frequency, and nature of use. Such testing programs have
been influenced strongly by requirements and guidelines
for testing regulated chemicals.
4. A substantial effort is maintained by industry to con-
trol or regulate the purity of raw materials or intermed-
iates used in chemical synthesis. The presence of toxic
or potentially toxic impurities is also tightly controlled
once these have been identified.
5. The development of a simple, inexpensive, and reliable
toxicological screen is needed to bridge the gap between
single-dose acute studies and long-term repeated dose
studies to serve as an early warning of potential hazard.
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6. Not unique to this survey is the observation that re-
liable and less time consuming systems to detect multi-
generation and carcinogenic effects are highly desirable,
7. Tests that might be most predictive in the detection
of potential mutagenicity and environmental hazard are
still being developed. Such development needs to be en-
couraged by moving cautiously in establishing rigid pro-
tocols or guidelines.
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II. RECOMMENDATIONS
In order to provide the maximum protection to man and his
environment against the hazardous use of both new and exist-
ing chemicals on the one hand, and on the other to minimize
the financial burden to the manufacturer and eventually the
consumer and taxpayer, it is recommended that:
?- Screening methods be developed that are
inexpensive and reliable as early warning
tests for potential long-range or subtle
hazards. These would bridge the gap be-
tween single-dose and long-term (approach-
ing lifetime) and multigeneration repro-
duction studies.
- Development of less expensive and time
consuming tests for carcinogens be en-
couraged.
Latitude be permitted in test protocols
for those areas not yet well standardized,
particularly
o Mutagen tests
o Environmental damage tests
Establishing rigid protocols for these
areas at this time would tend to inhibit
additional basic research.
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- When a manufacturer is required to file
information under a Toxi|c Substances Con-1-
trol Act, each filing ibe accompanied by
an updated outline of jcompany procedure
and policy in assessing toxicological
and environmental hazards. There can be
no greater safeguard than a thoughtful
review by competent personnel abreast of
the current state-of-the-art.
A mechanism be devised to signal the need
for expanded toxicological and environ-,
mental testing. Such a mechanism should
take into consideration
o Gross amounts produced
o Maximum and mean concentrations re-
sulting from use at both normal and
abuse levels *
o Chemical stability and degradation
products
o Currently known toxic properties
Registration at definite interval^ could
require such an analysis be made for each
new filing.
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Ill. INTRODUCTION
Of the new chemical entities that are being developed to
the point of commercial products, the majority are regu-
lated by existing Federal legislation. Regulations gov-
erning the introduction to the market place of food addi-
tives, pesticides, drugs, medical devices, and cosmetics
are well known. Legislation regarding the transportation
of hazardous substances, the safety of household substances,
the safety of surface finishes, and flame-retardant require-
ments has also resulted in Federal regulations for the
evaluation of the toxicology of many chemicals or chemical
formulations. Finally, legislation designed to protect
the factory worker is increasing the regulation of chemi-
cal intermediates. Experience with these regulations will
effect profoundly any new toxic substances controls.
In spite of all of the existing regulatory practices with
respect to the toxicological and environmental evaluation
of chemicals, some chemicals can be identified that are
not regulated or are inadequately regulated to protect
both man and his environment.
Protection is needed in some cases against product misuse,
in some cases against overuse, and in a few cases against
any use that would expose man or his environment to apprec-
iable quantities.
Examples of chemicals that fall within the definition of
this study are:
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o Industrial chemicals ordinarily used in the produc-
tion of other chemicals, formulations, or polymerics
o Industrial solvents, dry cleaning liquids
o Propellants
o Fuels
o Flame retardants
o Heat transfer materials
o Pigments other than for foods, drugs, and cosmetics
o Fertilizers
The Toxic Substances Control Act when finally enacted is
intended to cover these chemical entities whether currently
in use or being introduced for the first time. To prepare
regulations for the orderly enforcement of this legislation
requires a knowledge of the practices being used currently
within the industry to assess potential toxicological and
environmental hazards of the use of such chemicals.
The purpose of this study then was to establish, as it
exists in the industry, the current state-of-the-art for
the assessment of potential hazards to man and the en-
vironment resulting from the introduction of new or exist-
ing chemicals. Of necessity this has involved the com-
parative evaluation of screening test methods, policies,
and procedures used. To accomplish this objective, a
sampling of the major chemical industries was undertaken
and information based upon site interviews, interviews
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with key personnel, public information documents, publi-
cations in the scientific literature, and activities of
several trade associations was compiled. From this com-
pilation, the ensuing evaluation has been prepared.
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IV. METHODS EMPLOYED
SAMPLE SELECTION PROCESS.
Selection of Organizations to be Considered;
As a basis for a good cross section of the chemical indus-
try, the Standard Industrial Classification four-digit code
was studied. From this review 40 four-digit classifica-
tions were chosen that from their descriptions appeared
to include those companies that had the capacity for pro-
ducing chemicals subject to the Toxic Substances Control
Act. These classifications are as follows:
3312 Blast furnaces and steel mills
2911 Petroleum refining
2841 Soap and other detergents
2819 Industrial inorganic chemicals, NEC
(Not Elsewhere Classified)
2824 Organic fibers, noncellulosic
3339 , Primary non-ferrous metals, NEC
2812 Alkalies and chlorine
3334 Primary aluminum
2823 Cellulosic man-made fibers
3241 Cement, hydraulic
2681 Gum and wood chemicals
2821 Plastics materials and resins
3292 Asbestos products
2851 Paints and allied products
2813 Industrial gases
2871 Fertilizers
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2873 Phosphates (transferred from 2874)
2842 Polishes and sanitation goods
2893 Printing ink
2895 Carbon black
2816 Inorganic pigments
2899 Chemical preparations, NEC
3255 Clay refractories
3341 Secondary nonferrous metals
2865 Cyclic intermediates and crudes
.2818 Industrial organic chemicals, NEC
2992 Lubricating oils and greases
2843 Surface active agents
2891 Adhesives and gelatin
2951 Paving mixtures and blocks
2822 Synthetic rubber
3251 Brick and structural clay tile
3253 Ceramic wall and floor tile
2892 Explosives
3333 Primary zinc
3332 Primary lead
2952 Asphalt felts and coatings
2999 Petroleum and coal products, NEC
3259 Structural clay products, NEC
3331 Primary copper
A listing of all companies by each SIC code number was ob-
tained from Dun & Bradstreet and was computer sorted to ob-
tain the name of the major one or two companies for each
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classification. This listing was cross-checked against
rosters of professional societies or attendees at scientif-
ic meetings that identified industry supported personnel
dealing in the assessment of toxicological and environmental
effects of chemicals. An additional 23 companies were so
identified. It is believed that the resulting 62 companies
so selected perform a significant industry role in the
assessment of the toxicological and environmental effects
of chemicals both regulated and nonregulated. Both direct
and indirect interaction with trade associations such as
MCA (Manufacturing Chemists Association), PMA (Pharma-
ceutical Manufacturers Association), CTFA (Cosmetic Toiletry
and Fragrance Association), and standards groups such as
ASTM (American Society for Testing and Materials) have pro-
vided additional validation of the above selected industrial
samples.
Initial Contact Procedure;
The initial contact with each company was made by telephone
to an individual that in most cases had professional quali-
fications as a toxicologist or industrial hygienist. The
purpose of the study to be undertaken was explained and a
request made for each company so contacted to participate
in the study. Nearly every company contacted expressed
willingness to cooperate (in some cases subject to review
by general counsel or management).
Follow-up Requests;
Exceptions to cooperation was based on the claim that the
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company produced no chemicals subject to the pending legis-
lation. One company agreed at first to cooperate but later
declined on the basis that their information would be for-
warded directly to EPA. Since the personnel involved are
personally known to the writer of this report, there is no
reason to believe otherwise.
Selection of Final Participants;
Because of federal regulations restricting the number of
industrial companies to be studied in depth to nine, a list
of 18 companies, a primary and an alternate in each of nine
categories, was selected from those companies that had
agreed to cooperate. This selection was made during a con-
ference with the Government Project Officer and other EPA
personnel. The nine companies whose personnel were inter-
viewed for this report are:
Hercules, Inc.
Exxon Corporation
Proctor & Gamble Company
E. I. du Pont de Nemours & Company, Incorporated
Eastman Kodak Company
Minnesota Mining and Manufacturing
Shell Oil Company
Hoffmann-LaRoche Inc.
Allied Chemical Company
The writer of this report is very appreciative of the
time the above companies made available for the study. It
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is only regretted that it was not possible to take advan-
tage of all offers of cooperation that were extended. This
was not possible because of the OMB restriction previously
mentioned. On the other hand, while each company provided
procedures unique in detail, the overall policy of each
company with respect to toxicological and environmental
assessments was aimed at objectives quite uniform within
the industry. In the companies studied, these objectives
were diligently pursued and matters of product safety had
the attention of upper management. Such diligence and
"attention may not be representative of the entire industry.
VISIT TO SITE AND INTERVIEW.
Preparation of Outline for Interview;
Based upon knowledge obtained over many years' experience
in both government and industry in investigations of prob-
lems related to the toxicity of chemicals, it was realized
that many factors over and above experimental protocols must
be considered when evaluating potential environmental and
toxicological hazards. In order to obtain information on
all important factors, an outline was devised to be used in
each interview. After the initial one or two interviews,
the outline was followed from memory. Information sought
included the following:
o Decision making processes related to toxico-
logical and environmental assessments.
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o The commitment made to such assessments in
terms of personnel, dollar value or product
development.
o Mechanisms for monitoring or controlling
marketed products.
o Criteria used to continue or abandon testing.
o Criteria used to limit product distribution
or warn users.
o. Specific test protocols.
o Criteria used to select appropriate test protocols.
o A listing of warning signs that would indicate
more experimental work was required.
o The effect of chemical impurities on safety
assessment data.
Conduct of Interview:
Meetings with company personnel and interviews were for the
most part informal, although in a couple of sessions a
representative from the legal staff of the company was pres-
ent. Care to avoid judgmental statements was made in order
to encourage the free and easy exchange of information. A
commitment was made to each company that specific procedures
utilized by that company would not be identified with the
name of the company in the final report. A few unique test
protocols were described.
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Collection of Available Documents;
In a number of cases, available published documents were ob-
tained and included items such as annual reports, product
information sheets, listing of company products, organiza-
tion charts, and publications from the scientific literature
by company personnel. In a couple of instances, hard copy
test protocols were provided. However, in most cases such
test protocols were said to be "standard" procedures fol-
lowed by all investigators.
ORDERING AND SUMMARIZING DATA FOR EACH COMPANY.
Following each site visit, the notes taken by each inter-
viewer (usually two) along with written material, docu-
ments, and ancillary information were used to compile an
individual "case history" for each company with respect to
toxicological and environmental assessment of chemicals.
These individual "case histories" form the basis for this
report, augmented to some extent by ancillary information
as described below.
USE OF ANCILLARY INFORMATION.
A number of other organizations are in various stages of
collecting, writing, or standardizing test protocols or
guidelines for the toxicological and environmental assess-
ment of chemicals. The ASTM has established Committee
E.35 to produce standard protocols for pesticides. PMA
in cooperation with FDA is attempting to establish guide-
lines for drugs. The Society of Toxicology along with the
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Association of Official Analytical Chemists (AOAC) has a
joint project to establish general guidelines for toxico-
logical testing.
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V. EVALUATION OF THE PRESENT INDUSTRY PRACTICES
DECISION MAKING PROCESSES.
Decisions with respect to toxicology and environmental ef-
fects of chemicals include resolutions of the following
questions:
- When to initiate laboratory testing.
How extensive should the laboratory testing be.
What restrictions should be placed on the use
of the chemical as a result of the test.
When should more extensive testing be done.
o What criteria can be set up to determine
the time for expanded investigations.
o What safeguards can be instituted to
assure that the need for additional
testing will be signaled at an ap-
propriate time.
What impurities may be permitted in the chemical
and at what levels must these be held.
What new applications will require a new review
of the toxicological and environmental prop-
erties of the chemical.
In all companies cooperating in this effort, these de-
cisions are made by professional toxicologists who would
be qualified on the basis of advanced degrees and from
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several to many years of experience in toxicology, bio-
chemistry, or medicine. In decisions involving major
expenditures or massive chemical exposures, consultants
often are used to provide additional perspective. While
such decisions are subject to review and must be defended
by the toxicology staff, various strictures prevent over-
riding or ignoring them by other elements of company
management and include:
Formal corporate policy and procedures with
a written sign-off by responsible personnel.
- Sign-off by legal counsel.
Relationships with liability insurance carriers.
- Making product managers or profit center managers
accountable for any product liability.
The practices found in the companies cooperating in this
effort are representative of those seen elsewhere in
companies both large and small that engage the services
of a contract toxicology and analytical laboratory.
Decisions Regarding New Products; Decisions regarding new
products are made initially when a chemical becomes avail-
able to the Product Manager, usually from Central or
Division Research, or in some cases from "outside acquis-
itions." Typically, several candidate materials will be
available and the very first, decision will be that of the
best choice of the candidates. Depending upon intended
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end use of the chemical, toxicological and environmental •
properties will be a greater or lesser factor in the final
choice of the compound to be developed. Ordinarily, these
decisions follow joint agreements made by marketing, en-
gineering, production, product application, and safety
personnel.
Once the most promising candidate compound is selected,
laboratory testing programs are established, the extent
of which is determined by:
- Proposed end use - industrial chemical, con-
sumer product, structural components, energy
transfer
Extent of human and environmental exposure -
minor, visible, massive
- Duration of exposure - infrequent, occasional,
often, daily
Relationship to chemical structures having
known toxicological or environmental profiles
Chemical properties - stability, metabolic
products, degradation products, impurities,
binding characteristics
Physical properties - volatility, radiation
adsorption, particle size
These considerations lead to several possible levels of
laboratory and field experimentation as will be discussed
later.
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Decisions Regarding Existing Products; Decisions regard-
ing existing products with respect to assessment of toxi-
cological and environmental properties are the least well
defined of all decisions in safety evaluation. Such de-
cisions, when made, are usually reactionary. A company
reacts to:
Consumer complaints.
Industrial customer complaints.
Adverse publications in the scientific literature.
- Governmental regulatory action.
Unions, employees.
Seldom does the industry anticipate these complaints or
adverse findings since no machinery has been set up to
trigger the need for additional investigations. As a nat-
ural progression in a free market place, the extent of use
of an available chemical will remain constant, diminish,
or increase. If the use remains constant or diminishes,
there is no requirement for additional toxicological or
environmental evaluation unless the use pattern changes
substantially. The increasing use of a chemical is the
primary problem producing situation. A current example of
such a problem is vinyl chloride. Its production in the
United States has increased from 321 million pounds per
year in 1952 to an estimated 5.3 billion pounds per year
in 1973 (1). At some point in time, this product growth
should have triggered substantially increased attention to
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its toxicological and environmental properties. This did
not occur. It was only after some adverse publications in
the scientific literature that the American industry began
to react. Even then, the reaction was not aggressively
pursued so that at this writing additional adverse findings
in Europe have appeared before positive industry action in
America has been made public (2).
Compounds that fall within the category of existing products
have provoked the greatest present concern to an increas-
ingly consumer oriented public. The polychlorinated bi-
phenyls, fluorocarbons, DDT, vinyl chloride, asbestos, and
certain halogenated solvents are such examples. The use
in each case began with a few thousand pounds which could
be shown to be safe. Current usage has grown in each
instance to millions to billions of pounds. This increased
usage should have triggered within the industry the develop-
ment of new toxicological and environmental testing programs,
This did not occur. It remained for scientists in the
academic community to become concerned and to seek Federal
support for programs to assess the possible effects of the
massive use of such chemicals. The possibility that there
exists a number of other currently marketed chemicals that
have been inadequately studied in relationship to their
present level of use is almost certain.
Problems leading to the lack of adequate decision making
in the area of existing products include:
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Absence of corporate policy requiring reexamina-
tion of toxicological and environmental effect
profiles at predetermined production or use
levels.
Loss of proprietary position because the patent
protection period has expired. Many companies
may then share responsibility.
Absence of industry-wide monitoring programs
that would signal the need for more attention
to toxicological and environmental impact.
Failure of primary producers to monitor changes
in user applications.
- Failure of regulatory agencies to recognize in-
creased areas and extent of use. Again using
the vinyl chloride example, uses were condoned
that far exceed industry's own evaluation of
the toxic potential (3).
Decisions Regarding Product Changes; Decisions regard-
ing product changes have been carefully considered by all
companies cooperating in this project. In fact, a major
effort in each company is in existence to evaluate, moni-
tor, and/or approve of any changes in sources of raw
materials, specifications for feed stocks, or new methods
of synthesis. The need for such attention has been
highlighted by the existence of the dioxins in 2,4,5-T(4)
and bis-chloromethyl ether contamination of chloromethyl
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methyl ether (5). The dioxins are among the most toxic
substances known and are teratogenic at very low levels
of exposure. The presence of bis-chloromethyl ether may
be responsible for cancers reported in workers exposed
to chloromethyl methyl ether. Uncertainties in his-
toric supplies and spot shortages of material have made
it necessary to consider alternate sources of raw materials
and intermediates. These new sources offer supplies that
invariably differ from traditional supplies with respect
to impurities. These may be various heavy metals and/or
organic substances with known toxic properties. It then
becomes a task for the industrial toxicologist to determine
whether or not the new source is satisfactory in light of
previously published information. Obviously, this can be-
come a formidable effort in multiproduct companies. Fre-
quently, it is necessary to consider not only the intrinsic
toxicity of the impurities, but also the influence these
impurities may have on:
Catalytic degradation, hydrolysis, or conversion.
- Production of new impurities in synthetic processes.
- Physical properties of the finished product.
These problems are particularly important in the production
of pharmaceuticals, polymers, and pesticides.
DEVELOPMENT AND USE OF SPECIFIC EXPERIMENTAL METHODOLOGY.
From interviews with company toxicology personnel and re-
views of stated policy, four levels of experimental test-
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ing have been identified that correspond to the same num-
ber of levels of exposure of either man or his environ-
ment. These levels are derived from consideration of
length of exposure, extent, avoidable or not, numbers of
people at risk, and the portion of the environment exposed.
While there may be some overlap, depending upon unique
conditions of exposure for a particular chemical, testing
procedures may be grouped as follows:
Level I Exposure;
This originally occurs when the chemical is still in lab-
oratory or pilot plant production where a limited number
of persons may be exposed. Later, this level also includes
the single or infrequent chemical contact through accidental
splash, spill, or container rupture. Trivial exposures
such as might be experienced by laboratory personnel
developing potential end uses are considered here. For
this level, the studies usually conducted are:
Single Dose Studies Irritation Studies
o Oral LD5Q o Eye, mucous membrane
o Emetic potential o Dermal
o Inhalation LD5Q o Corrosiveness
o Dermal
Level II Exposure ;
As more persons become involved in the production or indus-
trial use of the chemical where the exposure rates are low
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or where high concentrations can be avoided, or where pre-
cautionary procedures can be employed, toxicological in-
vestigations are expanded to include:
10- to 90-Day Studies By Sensitization Studies
o Oral o Dermal - guinea pig
o Inhalation o Dermal - man
o Dermal routes o Inhalation - guinea pig
Environmental Behavior
o 96-hour fish LD5Q's
o 5- to 7-day bird toxicity
o Half-life determinations in water, soil, etc.
o Biological or chemical oxygen demand (BOD, COD)
Level III Exposure;
As more and more individuals become exposed through oc-
cupational, hobby, repair, or incidental chemical contact,
this level has been reached. This includes through the
hobbiest, repairman, or craftsman some limited consumer
exposure. Frequent low-level or occasional high-level
exposures of substantial populations usually are also in-
cluded within this definition. Studies progressively pur-
sued are:
Teratology
Mutagenicity
Animal or human metabolism
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Environmental
o Biodegradation
o Metabolic or decomposition products
o Transport mechanisms
Level IV Exposure;
This level is usually defined as occurring when use has
become so widespread that nearly everyone is exposed to the
chemical or use is such that exposure to it in small amounts
becomes unavoidable. Other types falling within the cate-
gory are frequent high-level or general consumer exposures.
Additional studies conducted for this level are:
Reproduction
Carcinogenic
Prolonged or life-time
Environmental
o Disposition
Sewage treatment
Incineration
Landfill
p Food-chain accumulation
o Long-term aquatic/wildlife
Laboratory and field testing usually proceed stepwise
from Level I through Level IV, depending upon the degree
of exposure to the chemical expected. At each level of
exposure described above, some estimate of accidental or
abuse usage must be projected. A degree of accidental
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or abuse exposure should be allowed for by employing safety
factors applied to data from laboratory experiments.
While the degree of exposure estimated by one toxicologist
will vary from that estimated by another, there was quite
good agreement among those interviewed for the classifica-
tions, as defined above. In the event of doubt about which
exposure level best describes the end use of a chemical,
the testing procedures to be employed for the next higher
category may be utilized.
Because of existing Federal regulations regarding the trans-
portation and labeling of chemicals, nearly every new chem-
ical entity produced in commercial quantities will undergo
applicable Level I testing procedures. What frequently
happens, however, is that the use of a chemical grows so
gradually into a Level II category that no one thinks of
conducting Level II studies. This pattern repeats itself
through Level IV. It should be noted also that very little
or no experimentation is conducted on the environmental
effects of a chemical in the Level I category. This lack
of environmental studies is in keeping with the limited or
restricted use that can occur within the definition of
Level I exposure.
Typical protocols are appended. However, as a guide a gen-
eral description is given here.
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Level I;
Single Dose Studies - Single dose studies are conducted in
one or more species in decreasing order; rats, mice, rab-
bits, guinea pigs, dogs, cats, monkeys, domestic (economic)
animals. Several dose levels are employed using one to ten
animals per sex per dose so that an LD5Q with 95% confidence
limits can be calculated at least for a rodent species. The
most applicable route of administration is used, most often
oral, dermal, or inhalation. If dogs, cats, or monkeys
are used, the emetic potential will be apparent. Animals
are observed for seven or more days following exposure and
animals that die and representative survivors at the end
of the observation period are examined for signs of organ
damage at necropsy. Close attention to pharmacologic and
toxic signs in such experiments is stressed.
Irritation Studies - Irritation studies are most frequently
conducted in the eye and on the skin of the rabbit. For
some purposes, the guinea pig may be immersed in the mate-
rial under test. Also, a specific evaluation of the cor-
rosiveness of the material to the trachea or hamster cheek
pouch may be needed where exposure to children becomes a
possibility. From three to ten animals (most commonly six)
are used for each exposure condition or concentration. Some
scheme of describing in a quantitative fashion any reaction
seen is ordinarily employed.
Level II;
Repeated Dose Studies - Repeated dose studies of 10 to 90 •
27
-------
days in duration are usually conducted in rats for oral
and inhalation routes of administration, and in rabbits for
the dermal route. Dogs, monkeys, or cats are also employed
when a non-rodent test species is desired. At least four
groups of animals are used with one group being an untreated
control and the remaining groups given dose levels ranging
from no (or marginal) effect to a maximum tolerated level
showing definite signs of toxicity. Group sizes are most
frequently 3 or 4 of each sex for non-rodents, 10 to 15 for
rats, and 4 or 5 for rabbits. Studies of 10 to 14 days
duration for screening purposes utilize smaller groups.
Parameters for study include weight changes, hemograms,
clinical chemical tests, changes in behavior, and appear-
ances of the eyes, coat, and mucous membranes. At termina-
tion of the studies, histopathological examination of the
organs and tissues is made in order to determine most
probable target organs.
Sensitization Studies - Sensitization studies are conducted
first in guinea pigs and secondly in man to estimate the
potential of the chemical to elicit an eczematous sensiti-
zation following repeated exposure. Ten to 25 guinea pigs
and 50 to 200 people are most often used per compound.
Environmental Impact - Environmental impact studies are
first encountered in Level II but probably more often in
Level III. These studies are confined to screening studies
using Daphnia, fingerlings of available local fish, and
28
-------
Chlorella. Such screening studies are not used industry-
wide and tend to be company specific and remain basically
exploratory in nature. Standard protocols that are followed
consist of: .
o BOD
o COD
o 96-hour LC50's in one or more species of fish
o A 5- to 7-day toxicity study in one or two
species of birds such as bobwhite quail and
mallard ducklings.
These protocols are described in ASTM publications and in
EPA guidelines for pesticides.
Chemical studies may also be made that are helpful in pre-
dicting environmental impact and these include:
o Oil/water distribution coefficients or, more
often, solubilities in water and a variety of
nonaqueous solvents
o Hydrolysis rates
o Stability studies using exaggerated conditions
of heat, humidity, and/or light under acid,
alkaline, and neutral conditions.
Level III:
Level III studies generally include several of the follow-
i
ing:
29
-------
Teratology Studies - These studies are done most often in
rats but also in hamsters, mice, and rabbits, and on rare
occasions in other species such as dogs, monkeys, or domes-
tic animals. The basic study involves administration of a
test compound during the gestation period corresponding to
the days of organogenesis. Fetuses are removed usually
one or two days prior to normal birth. Some of these are
cleared and stained in order to permit examination of skel-
etal development. The remainder are preserved to allow
subsequent examination of soft tissues for any abnormal
development. Groups of 20 or so pregnant females for
rodents and 5 to 10 for non-rodents are employed using
two or more dose levels, a control level and sometimes one
level of a reference compound.
Mutagenicity Studies - Mutagenicity studies are beginning
to be employed with greater frequency in the investigation
of new chemicals. A lack of general consensus as to the
proper methods for such assessment has hampered the develop-
ment of effort in this area. Methods most frequently em-
ployed include:
o Some version of the Bateman Dominant Lethal
test, using either mice or rats.
o A version of the host-mediated assay.
o A system using bacteria, an in vitro cell sys-
tem, or some small organism that multiplies
rapidly.
30
-------
Versions of such test protocols are described by Hollaender
(6) .
Animal (Human) Metabolism - Animal (human) metabolism
studies are being employed frequently at this level partic-
ularly since at this point in the exploitation of a chemical
it becomes important to compare the metabolic disposition
in laboratory animals, in the environment and in man. Such
studies provide the basis for refining estimates of the
levels of exposure that may be permissable. These metabo-
lism studies generally follow a standard pattern progres-
sing as follows:
o Absorption, and excretion in animals
(possibly including man)
o Metabolic fate in the animal
o Metabolic fate in plants or other biotic
compartments of the environment.
o Identification of metabolites, breakdown
products, and sites of accumulation in
both animals and in the environment.
fcnvironmenta1 Studies - Environmental studies for Level
III include those described for Level II if they have not
yet been conducted and in addition:
o Biodegradation in receiving waters, soils
o Soil and water movement of transport
31
-------
o Identification of metabolites or decom-
position products if not already achieved
as noted above.
For most of these studies, standardized protocols are
still under development. However, a few selected proto-
cols have been described by ASTM (7) such as for the bio-
degradation of detergents. Sound analytical chemical pro-
cedures otherwise are followed in attempting to answer the
questions regarding fate in the environment and the ki-
netics involved.
Level IV;
Level IV studies generally are employed only as a reaction
by the industry to some adverse finding or publicity. A
number of monomers in addition to vinyl chloride are under
study or a new review. A number of industrial solvents
that have been in use for years are receiving renewed at-
tention. One exception to this generalization was the mas-
sive amount of work conducted on NTA (sodium nitrilotri-
acetic acid) prior to any massive human and environmental
exposure (8,9). This level of testing for NTA indicates
a recognition of the potential adverse effects of the
introduction into the environment of massive quantities of
a new chemical. The lesson of the phosphate detergent
builders was not ignored. The additional studies usually
considered in Level IV investigations include all of the
following where applicable:
32
-------
Reproduction Studies - Reproduction studies usually are
conducted in rats for three generations. Groups of 20
animals of each sex will be exposed by the appropriate
route to various levels of the compound beginning 60 days
prior to mating and through gestation and lactation of
second litters. Selected animals from second litters
will then be used as parents of the second generation.
This will be repeated through the second litters of a
third generation.
Carcinogenic Studies - Carcinogenic studies are normally
conducted in both rats and mice by the appropriate route
for the approximate life span of the strains used (usually
18 to 24 months). Two or three dose levels are employed
with 35 to 50 animals of each sex per group. Historic
data along with an equivalent or even larger group of
control animals are required in order to lend validity to
the results obtained. Gross and histological examination
of the tissues of all animals, both surviving and non-
surviving, must be made to avoid possible experimental
artifact.
Prolonged or Lifetime Studies - Prolonged or lifetime
studies may be employed using non-rodent species to detect
chronic organ damage other than cancer. These studies
generally extend over 6 to 24 months. In rare instances
studies approaching lifetime in dogs and monkeys are, or
have been, undertaken. The numbers of animals per group
33
-------
are usually three or four of each sex. Study parameters
include growth, biochemical, hematological, neurological,
behavioral, histopathologic, and eye changes. Details of
these study parameters are listed in typical protocols
appended. Some exceptional studies have used larger num-
bers of animals. Birth control drugs are studied for
seven years in dogs, 10 years in monkeys, with group sizes
of 16 of one sex.
Environmental Disposition Studies - Environmental disposi-
tion studies include an evaluation of the consequences of
the disposal of the chemical, or materials containing the
chemical, by one of the avilable methods:
o Waste water treatment systems
activated sludge
septic
oxidation ponds, trenches, filters
o Incineration
o Landfill
o Composting
Commonly employed protocols are not available so that such
studies fall more nearly within the category of basic re-
search. Sound analytical chemistry and thoughtful experi-
mental plans are obviously of paramount importance.
Movement Within the Environment - Movement within the en-
vironment or mobilization of previously bound or fixed
34
-------
materials as a consequence of the chemical is also studied
at this level. Evidence on the following points is sought:
o Possible buildup through food chain
magnification.
o Bioaccumulation by specific plants and
~>
aquatic organisms.
o Mobilization of existing toxic substances
that have been bound or fixed in bottom muds
or in poorly drained soils. These are most
often inorganic substances.
MONITORING SYSTEMS:
One critical area that uniformly receives attention by the
companies cooperating in this study is that of monitoring
their marketed products from the standpoint of safety.
This takes the form of:
Consumer Complaints Handling - These arise in several ways
and all are handled by the Toxicology Staff:
o Telephone "Hot Line," a nationwide 800 area
code number which is attended 24 hours a day,
seven days per week, by someone sufficiently
trained to give advice or who knows where to
obtain it for any emergency related to a
chemical exposure.
35
-------
o Complaint letters containing any remarks re-
garding ill effects to plants, animals, or
man.
o Complaints from marketing or sales personnel—
usually from industrial customers, rather than
individual consumers.
»
Marketing Liaison on New Applications - Marketing liaison
on new applications is generally managed in either a for-
mal way in a few companies or informally in the remain-
ing companies, by designating a "Toxicology Liaison" indi-
vidual in each operating division. This person is charged
with the responsibility of clearing any question regarding
toxicity or adverse environmental effect arising within
his division:
o First with the company toxicology and/or
environmental staff.
o Secondly with outside consultants or
advisors.
o Sometimes through inquiry with a trade
association.
o Often through inquiry with his professional
counterpart in another company.
Various built-in safeguards are established to assure that
such precautions are taken. Such safeguards include:
36
-------
o Insurance carrier review. Some product
liability insurance companies base rates
in part on toxicological information avail-
able.
o Charges to profit centers for product
liability when safety procedures have been
ignored or compromised. The profit center
may be a company division or a product line
production and sales group which has been
identified and held responsible for returning
a profit to the company.
o Review by legal counsel.
When to Expand Toxicological and Environmental Testing -
No company within those cooperating in this study has a
system that would automatically trigger an expanded test-
ing program. Some mechanism to bring about this kind of
attention to toxicological and environmental problems is
yet to be devised, based upon information gleaned in this
survey. A variety of factors have contributed to the
lack of development of such a system. These include:
o Loss of proprietary position and resulting
fragmentation of responsibility for the
product produced when patents expire.
o Growth of product use has failed to alert
responsible personnel in the absence of any
37
-------
fixed company guidelines. To establish
fixed guidelines would involve some measure
of acute, chronic, and incidental or minor
exposures, along with an evaluation of the
degree and nature of toxic or environmental
sequelae. At this point in time no such
formula has been advanced, much less tried
empirically.
o Because of the foregoing problems, the total
extent of use of a chemical ceases to be
under review of a single individual. Con-
sequently, the fragmented use of such a
material, while being observed in individual
situations by competent personnel, lacks
the impact of a knowledge of the total ex-
posure. The United States Tariff Commission
compiles production data on chemicals that
could be used as part of an alerting mech-
anism. . . .
o Also because of the foregoing situations,
the availability of new methods for the
assessment of toxicological and environmental
effects of chemicals are riot followed up by
individual company action.
CHEMICAL PURITY:
While specifications for chemical purity have not been
discussed in connection with company policy or testing
38
-------
programs, it is recognized throughout the industry that
the chemical identity and purity of any chemical used
in toxicological and environmental studies must be
specified carefully. Consequently, any study being
undertaken currently will be reported in terms of a
particular batch, or mix of batches, for which there are
available chemical analyses showing:
o Content of active ingredient
o Moisture
o Organic solvent residual
o Amount and nature of impurities
o Heavy metal content.
Batches of chemicals exceeding these specifications are
not covered by the toxicological and environmental experi-
mental data obtained.
Industry generally has become very sensitive to the
problem of impurities, especially since the 2,4,5-T epi-
sode and a few less publicized examples, such as the
content of beta-naphthylamine in alpha-naphthylamine.
COSTS FOR CONDUCTING TOXICOLOGICAL AND
ENVIRONMENTAL HAZARDS ASSESSMENT:
Appended to this report is a copy of a paper (10), which
reports a survey of costs for nearly all of the test
39
-------
methods described for toxicology. Several of the labora-
tories that participated in the survey have also partici-
pated in the survey reported here. In addition, a number
of "independent" laboratories participated in the survey
so that reported costs are quite well documented. The
cost survey was completed in 1972. Consequently, a factor
at least equal to the intervening increase in the cost
of living index must be applied to these figures to make
them applicable today.
Excepting costs of tests on birds and aquatic life con-
tained in the above cited survey, no good estimate of
costs were possible for the various environmental test
procedures.
40
-------
VI. IDENTIFICATION OF PROBLEM AREAS
IN TOkE.C SUBSTANCES EVALUATION
NEW PRODUCTS:
The majority of new chemical entities that become commer-
cially available are not examined beyond Level I tests.
The degree or extent to which man or his environment is
exposed to such chemicals does not warrant more extensive
testing and by the same token the sales volume would not
support the costs of more extensive studies. Unfortunately,
Level I tests are based upon single or acute exposures and
give no clue or warning of the more subtle effects that
can follow repeated low-level use. There is a need for
a biological screening system that would be predictive
or would serve as a warning of possible long-term effects.
Such a system need not evaluate long-term effects, but
should be capable of signaling possible adverse effects
should the chemical enjoy increasing use. The cost of
such a test system should compare favorably with other
Level I tests.
A 5- to 14-day repeated dose LD5Q screen is used by two
companies in this survey, and by two other companies
outside the survey, in an attempt to guard against re-
peated dose "toxicological surprises." Regulatory re-
quirements for single dose LD-'s and irritation studies
41
-------
may have inhibited the development of more rational short-
term repeated dose studies.
When the projected level of use of a new chemical places
it into categories requiring more extensive testing,
there is currently available satisfactory methodology
to answer known concerns about possible toxicity and
environmental effects. These higher level tests, however,
are relatively expensive so that they will not be employed
until a satisfactory product profitability can be pro-
jected. Obviously, if inexpensive but reliable screening
tests could be developed, potential toxicological and
environmental hazards would be known at a much earlier
time.
EXISTING PRODUCTS:
By far the most serious deficiency in present programs
for the assessment of toxicological and environmental
hazards of a chemical is the lack of any mechanism to
trigger expanded levels of testing. The typical case
history of a chemical involves the production of rel-
atively small amounts of the chemical, usually less than
a tank car full. At this time, Level I toxicity tests are
the maximum justified. Availability of the chemical is
made known, along with its physical and chemical prop-
erties. I'f use of the chemical catches on within the
.next several years, and if the manufacturer can maintain
some proprietary position, it is likely that Level II
42
-------
tests will be conducted, but there is no assurance of this.
With but a few exceptions, any testing beyond Level II will
be done as a reaction to: some problem and not as a planned
affirmative action. The exceptions have been investi-
gations on chemicals to replace those now known to be
troublesome either because of newly discovered toxic prop-
erties or potential damage to the environment.
43
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REFERENCES
1. Rowe, V. K. Testimony, Public Hearing on Vinyl
Chloride. Occupational Safety and Health Adminis-
tration, Department of Labor, Washington, D.C.,
February 15, 1974.
2. Maltoni, C. G. Lefemine, and L. Gualano. Preliminary
Report on the Carcinogenicity Bio-Assays of Vinyl
Chloride, Institute di Oncologia "F. Addarii" and
Centro Tumori, Bologna, Italy, February 11, 1974.
3. Torkelson, T. T., F. Oyen, and V. K. Rowe. The Toxi-
city of Vinyl Chloride as Determined by Repeated Ex-
posure of Laboratory Animals. American Industrial
Hygiene Association Journal, 22, 5, October 1961.
4. Cooper, P. Articles of General Interest. Con-
tinuing Uncertainties Over 2,4,5-T. Food and Cos-
metics Toxicology, 12, 3, pp 418-421, June 1974.
5. Drake, J. J. P. Articles of General Interest.
Questions of Haloether Carcinogenicity. Food and
Cosmetics Toxicology, 12, 4, pp 551-552, August
1974.
6. Hollaender, A. Chemical Mutagens. Principles and
Methods for Their Detection, Volumes 1 and 2, Plenum
Press, New York, New York, 1971.
44
-------
7. ASTM Standards. Industrial Water: Atmospheric
Analysis, Part 23, latest edition.
8. Anonymous. Articles of General Interest. Hope
for NTA? Food and Cosmetics Toxicology, 11, 4,
pp 674-677, August 1973.
9. Charlesworth, F. A. Articles of General Interest.
More Studies on NTA. Food and Cosmetics Toxicology,
!£, 4, 421-423, June 1974.
10. Gehring, P. J., F. K. Rowe, and Susan B. McCollister.
Toxicology: Cost/Time, Food and Cosmetic Toxicology,
11, 6, pp 1097-1110, December 1973.
11. The Fortune Directory of the 500 Largest Industrial
Corporations, Fortune, pp 230-260, May 1974.
12. Ball, R. The Secret Life of Hoffmann-LaRoche,
Fortune, pg 130, August 1971.
45
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VIII. APPENDICES
Page
A. Industrial Profiles of Companies Included
in Survey 47
B. Toxicology: Cost/Time 48
C. Protocols 62
46
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INDUSTRIAL PROFILES
OP COMPANIES INCLUDED IN SURVEY
Exxon
E.I. du Pont
Shell Oil
Eastman Kodak
Proctor & Gamble
MMM
Allied Chemical
Hercules
Hoffmann-La Roche
Annual (7)
No.
Toxicology
Effort**
•:s Billions
25.724
5.275
4.883
4.035
3.906
2.545
1.664
1.154
1.827*
Employees
137,000
118,400
32,000
120,700
47,000
78,900
33,600
24,100
39,000*
Inside Outside
-
++ ±
++ +
++ ±
++
++
++
++ +
* Projected estimates from Fortune Magazine, August
1971 (8)
** Toxicological effort related to experimental laboratory
work
47
-------
Fd Cosmet. Toxicol. Vol. 11, pp. 1097-1110. Pergamon Press 1973. Printed in Great Britain
Toxicology: Cost/Time*
P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
Chemical Biology Research, The Dow Chemical Company,-Midland, Michigan 48640, USA
(Received 27 June 1973)
Some type of evaluation of the toxicity of pesticides and the hazard of exposures there-
from has been carried out since the advent of pesticide use. Originally, pesticide manu-
facturers assumed it their moral responsibility to evaluate hazards and provide directions
for safe handling and use. This developed into a legal responsibility as well. Several legis-
lative acts have provided for pesticide regulation and all its ramifications including toxicity
and safety evaluation (McCollister, 1965; US Department of Agriculture, 1968).
The Federal Insecticide, Fungicide and Rodenticide Act passed in 1947 and amended in
1959 and 1964 requires registration of all economic poisons prior to distribution in inter-
state commerce. Economic poisons include a wide variety of agents, pesticides among them.
For registration to be obtained, data must be presented which demonstrate that the material
is safe when used as directed and effective for the purpose claimed on the label. The Act
also states that any residues remaining on food or feed must not exceed the established
tolerance level.
The provision for tolerances is the result of the Miller Amendment in 1954 to the Food,
Drug and Cosmetic Act. The original Food, Drug and Cosmetic Act, passed in 1906,
provided that a food should be deemed adulterated if it contained any added poisonous
or deleterious substances. The only exception was when such substances were required in
production or could not be avoided in good manufacturing practice. With passage of the
Miller Amendment, pesticide chemicals were officially recognized as essential to the pro-
duction of food, thus providing for the establishment of tolerances in raw agricultural
commodities. Tolerances are obtained by submitting to a governmental agency (formerly
the FDA, currently the EPA) a petition containing extensive data covering toxicity, meta-
bolism, residue levels, including an analytical method for determining residues on foods,
and finally a reasonable basis for supporting the safety of the proposed tolerance.
Another law that has greatly influenced the establishment of tolerances for pesticides is
the 1958 Food Additive Amendment to the Food, Drug and Cosmetic Act. This amendment
contained the Delaney Clause, which states that no food additive shall be deemed to be safe
if it is found to induce cancer when ingested by man or animals, or if it is found, after tests
which are appropriate for the evaluation of the safety of the food additives, to induce cancer
in man or animals. Many have interpreted this to mean that a zero tolerance must be ap-
plied to a pesticide found to produce neoplasms in animals regardless of dose, route of
administration or species.
'Presented at the 163rd ACS National Meeting, Boston, Mass., 9-14 April 1972; Symposium—Economics
of Pesticides: Current and Future.
1097
48
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1098 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLL1STUR
The purpose of this presentation is to elaborate on the toxicological studies that are
either required or recommended for registration of pesticides, including the establishment of
a residue tolerance on food crops. In particular, the costs and time required for completion
of the studies will be discussed. The values for cost and time were derived from information
supplied by the following toxicology laboratories: Food and Drug Research Laboratories
Inc., Dow Pharmaceuticals, Woodard Research Corporation, Mellon Institute, Hazelton
Laboratories, Gulf South Research Institute, Bionetics, Diamond Shamrock Chemical
Company, American Cyanamid, Eastman Kodak. Company, Eli Lilly and Company,
The Haskell Laboratory of E. I. DuPont de Nemours and Company, Vick Chemical
Division of Richardson-Merrell, The Dow Corning Corporation, the Chemical Biology
Research Laboratory of the Dow Chemical Company and Industrial Bio-Test Laboratories.
Each participant provided estimates, usually as a range, for conducting the various studies
described in a general protocol which was supplied. The time estimates included the time
needed to conduct the experiments and prepare the reports.
In the early 1950s, toxicology requirements for registration of a pesticide were generally
limited to data from single-dose oral administration and from a subacute study in which the
material was fed to rats for a period of 30-90 days, with measurement of a few indicators
of toxicity. Even in terms of today's inflated dollars, the cost of these studies would amount
to less than $10,000.
The picture is very different today. Three factors have contributed to more rigorous
and extensive toxicity testing. First the increased sensitivity of analytical methods for
detecting pesticide residues has increased the emphasis and concern over the possible
effects of low levels of pesticide residues heretofore undetected. Secondly, there has been an
increase in the sensitivity and number of techniques for monitoring signs of toxicity, such as
electron microscopy, biochemical methodology and radioisotopes and new test systems
such as teratology and mutagenicity testing. Some of these developments allow measure-
ment of different parameters of toxicity or of smaller deviations from normal in test
animals, while others merely add bulk. Thirdly, an increased awareness of, and concern for,
the impact of pesticides on the environment has led to more extensive studies on non-target
species in the environment, whereas previously, toxicology was primarily concerned with the
evaluation of hazard to man and to a species upon which the product was to be used.
The cost and time needed for completion of the acute toxicity studies required or recom-
mended for pesticide registration are presented in Table 1. These provide data for pre-
liminary evaluation of handling hazards as well as for characterization of the effects of
acute accidental or purposeful exposures to man and to parts of the environment.
The oral LD50 is the most common expression of toxicity obtained. This measures the
most drastic manifestation of toxicity—that of death. It is a statistically derived expression
of the single dose of a material that can be expected to kill 50% of the animals treated.
Various species may be used, such as rats, mice, guinea-pigs and rabbits. Along with pro-
viding numerical data, this test allows an opportunity for the observation of signs of toxi-
city in the surviving animals. In some cases, gross pathological and histopathological
examinations are performed to reveal the target organs. Acute oral toxicity data, including
the LDj0 and description of observed effects, are recommended on at least two species of
laboratory animal. The average low and high estimates of costs quoted by 14 laboratories
for these studies were $796 and $1230, with a mean of $1013. A period of 4-5 weeks is
required to complete the studies.
The dermal LD50 is also required. This is an estimate of the absorption of a material'
49
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TOXICOLOGY: COST/TIME
Table 1. Cost I time estimates for acute studies
1099
Test
Individual tests
Oral LD50
Dermal LD50
Inhalation LCSO
Eye irritation
Skin irritation
Avian oral LD30
Fish LC30
Crustacean LC50
Subtotal
Plus formulation
Plus 1 metabolite
No. of
quotes
14
14
12
14
14
8
9
3
Mean
(MS)*
1-01
0-65
0-89
0-26
0-26
1-88
0-58
0-62
6-15
12-30
18-45
Cost
Average ranget
(MS)*
5-16-7-11
10-32-14-22
15-49-21-33
Time required for
completion*
(wk)
6
12
18
•Thousands of dollars.
t Average of low estimates-average of high estimates.
JIf tests are run concurrently.
through the skin in acutely toxic amounts. Rabbits are the usual test animal. Other evidence
of toxicity besides death is also noted. The average range quoted for such a study was
$536-5758, with a mean of $647. This test requires 4-5 weeks for completion.
The inhalation LC50, also required unless it is not reasonable to expect such exposures
to be encountered, is the concentration expected to kill 50% of the test animals when the
material is inhaled for 1 hour. This study is conducted in the rat and costs ranged from
an average of $750 to $1020, mean $885. The study takes 4 weeks to complete.
In the required eye-irritation studies, the material is applied to the eyes of a rabbit and
observations as to the type and severity of damage are made at various times after exposure.
The effectiveness of washing the eye following application is also investigated. About 3
weeks are required to complete these studies, which range in average cost between $210
and $315, mean $262.
Skin irritation studies are also conducted on rabbits. The skin is clipped free of hair and
either abraded or left intact. Evidence of local irritation, which may vary from mild redness
to severe burn, is noted. The average range quoted for these studies was $217-$294, with
a mean of $255. Three weeks are required for completion.
Oral LD50 data are required in birds as well as laboratory animals. Bobwhite quail and
mallard ducks are recommended. These studies take 5-6 weeks to complete and range in
average cost from $1528 to $2225, mean $1876.
Acute toxicity studies in fish are also required. The estimates had an average range of
$525-$636, mean $580. Completion requires about 3 weeks.
Data on acute toxicity to other forms of aquatic organisms, such as shrimp and other
crustaceans, may also be required, costing on average between $500 and $633, mean $617.
Only three estimates were obtained. The studies take approximately 2 weeks.
The total cost, then, of these acute studies on a pesticide chemical would range on average
from $5162 to $7111, with a mean of $6135. At least 6 weeks would be required for com-
pletion, providing the tests were run concurrently. These acute studies are also conducted
5Q .
-------
1100
P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLL1STER
on the formulated product, and on major metabolite(s). Thus the total cost for acute
toxicity studies would average $18,405, and would require 18 weeks.
The subacutc toxicity studies are intended to evaluate the effects of relatively short-term
repeated exposures to pesticides (Table 2). Ninety-day feeding studies are required on the
parent chemical and on major mctabolite(s) in at least two species. These involve the
continuous administration of the test material to the animals, usually in their diets, at
several dose levels. One of the species has to be a non-rodent, and dogs are most often used.
The rodent species used is generally the rat. From 80 to 120 rats and from 16 to 24 dogs are
used in these tests and at least three dose levels are fed. There is also an untreated control
group. Observations are made regarding growth, food intake, appearance and demeanour,
. and mortality. Laboratory tests are conducted on urine for evidence of kidney damage and
on blood for untoward effects, such as anaemia. Various organ-function tests are conducted
Table 2. Cost/time estimates for subacute studies
Cost
= Time required for
No. of Mean Average rangef completion!
Test quotes (MS)* (MS)* (months)
90-Day /dogs: Parent chemical
Metabolite
90-Day /rats: Parent chemical
Metabolite
21 -Day dermal
14-Day inhalation
Reproduction: Quail
Duck
Total
Additional
14
14
14
14
11
10
4
4
17-4
17-4
12-8
12-8
7-0
6-1
12-4
14-8
100-7 88-7-112-3 13
13-2
'Thousands of dollars.
tAverage of low estimates-average of high estimates.
^Assuming studies on the parent chemical and metabolite are staggered.
§Studies on wildlife under field conditions, neurotoxicity and cataractogcnic potential.
by measuring blood levels of certain substances, such as urea and protein and the activity
of various enzymes. At termination of the study, selected organ weights are recorded. These
usually include the heart, Jiver, kidney, spleen, testes and brain. Gross and microscopic
examinations are conducted on approximately 30-tissues. Additional clinical studies may be
conducted, such as blood and brain cholinesterase determinations in the case of organo-
phosphate and carbamate insecticides. The objectives of these 90-day studies are to deter-
mine the nature of the effects related to ingestion of the compound and to define the mini-
mum dosage level at which the most sensitive criterion revealed an effect and the maximum
dosage at which no untoward changes were detected. The estimated cost of the dog studies
ranged on average from $15,795 to $18,967, mean $17,381. The rat studies cost slightly
less, averaging between $11,310 and $14,200, mean $12,765. Each study takes about 6
months for completion.
The 21-day dermal study consists of daily applications of the chemical to the skin of
rabbits, with observations made for evidence of absorption through the skin in toxic
51
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TOXICOLOGY: COST/TIME 1101
amounts. Body-weight gain and clinical and pathological parameters are evaluated. This
study was estimated to cost on average between $6380 and $7520, with a mean of $6940.
Completion requires 3-4 months.
A 14-day inhalation study in rats has been mentioned as sometimes desirable. It evaluates
the effect of repeated exposure to pesticides via inhalation, using parameters similar to
those discussed for other subacute studies. The average range of costs estimated by ten
laboratories was $5590-$6640, mean $6120. The time required for completion is 3 months.
Preliminary evaluation of the effects of pesticides on reproduction in two species of birds
is required. Bobwhite quail or pheasant and mallard duck are recommended. Egg produc-
tion, fertility, hatchability and survival are determined. These studies take from 6 to 7
months or longer to complete. Costs average between $10,000 and $14,750, mean $12,375,
for quail and between $12,500 and $17,000, mean $14,750, for mallard duck.
These subacute studies just described, including 90-day studies in two species on one
metabolite, range in average costs from $88,690 to $112,290, with a mean of $100,730. At
least 7 months would be required for the studies on the parent compound, assuming they
were run concurrently. The feeding studies on the metabolite(s) would be conducted later
and would take another 6 months. Other studies could be required, depending upon the
nature and use of the compound and the results of the studies just described. For example,
subacute studies on wildlife under field conditions would be necessary if indicated by LD50
data. Data from four sources give an average range of $2850-$3600, mean $3225. About
5 weeks are required for completion. A neurotoxicity study in chickens is required for
organophosphates, costing about $1000 (one estimate). Studies for evaluation of cataracto-
genic potential may be indicated and could cost $9000 (one estimate). The conclusion of
these additional tests would bring the average cost of subacute toxicity studies to $113,760.
Metabolism studies (Table 3) are conducted in an attempt to uncover the means by which
the animal deals with the chemical. These studies include investigation of which organ(s)
metabolize the material and the identification of major metabolites, absorption from the
intestine, storage in various tissues, and excretion in urine and faeces. Such studies use
some of the more recently developed analytical tools, notably radioisotopes and various
types of chromatography and spectrometry. Metabolism studies in rats and dogs are
required for pesticides. These investigations can vary considerably in extent, developing
into highly complex, expensive and time-consuming efforts. The scope of the studies usually
Table 3. Cost/time estimates for metabolism studies
Species
Rat
Dog
Man
No. of
quotes
7
7
Total
5
Cost
Average range*
(MS)f
9-6-20-4
9-2-20-3
. . . 18-8^0-7
17-5-24-7
Mean
(MS)t
15-0 .
14-7
29-7
21-1
Time required for
completion
(months)
3-12
3-12
3-121
1-3
•Average of low estimates-average of high estimates.
•(Thousands of dollars.
JIf run concurrently.
52
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1102 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
conducted on pesticides involves a period of 3-12 months and ranges in cost on an average
between $9600 and $20,433 for rats, mean $15,015, and from $9200 to $20,266 in dogs,
mean $14,733.
Carefully controlled studies may also be carried out to investigate the metabolism of the
pesticide directly in man and so allow for more meaningful comparisons with animal models.
Subacute toxicity studies must be completed first in order to evaluate the risk involved.
The experiments in man usually consist of administration of a single oral dose, followed by
identification and quantitation of the parent compound and metabolites in blood, urine
and faeces. The average of the range of estimates received from five laboratories was $17,500-
$24,666, mean $21,080. Any time from 1 to 3 months or more may be required for com-
pletion of investigations in man.
For some pesticides, studies in man may be conducted to determine a threshold level
which, if exceeded, will alter a sensitive biochemical activity. This is particularly appropriate
for cholinesterase inhibitors. The chemical can be administered to human subjects at low
dosages, and the blood cholinesterase activity can be measured as an indicator of physio-
logical response, with minimum risk to the test subjects. Time and money requirements
would vary considerably according to the particular design, but these studies may cost
$50,000 and could take a year for completion.
Studies revealing possible effects on reproduction or foetal development are required for
pesticide registration. Multigeneration reproduction studies (Table 4) are usually conducted
in rats, although mice, rabbits or guinea-pigs may also be used. The animals are maintained
on various doses of the test chemical throughout the study. The original parent group is
allowed to produce two litters and offspring from the second litter are in turn raised as
parents for the second generation, from which two litters are produced. A third generation
is usually derived in the same manner. Observations are made on each breeding trial
regarding fertility, survival of offspring, lactation and any evidence of abnormalities in the
foetuses. These studies in rats were estimated to average in cost from $33,360 to $37,460,
mean $35,410. Two years or more are required for completion. Studies in the other species
mentioned cost about the same.
Table 4. Cost\time estimates for multigeneralion reproduction studies
Species*
Rat
Mouse
Rabbit
Guinea-pig
No. of
qudtes
. 12
8
4
3-
Cost
Average rangef
(MS)J
33-4-37-5
22-4-27-4
26-5-38-8
27-3-36-6
Mean
(M8){
35-4
24-9
32-6§
31-8
Time required for
completion
(months)
24-25
24
32-35
30-35
*At least one required.
t Average of low estimates-average of high estimates.
^Thousands of dollars
§lt is anticipated that studies using rabbits would cost 15-20% more than those
using rats. The apparent discrepancy is attributed to the fact that laboratories
submitting the highest quotes for rats did not submit estimates for rabbits.
53
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TOXICOLOGY: COST/TIME
1103
Although the multigeneration reproduction test provides for some evaluation of tera-
togenic potential, separate, more specific, studies are recommended and are usually con-
ducted (Table 5). These studies involve administration of the test material in several doses
Table 5. Cost]lime estimates for teratogenicity studies
Species*
Rat
Mouse
Hamster
Rabbit
Monkey
No. of
quotes
12
9
5
10
4
Cost
Average range!
(M8)J
7-4-13-5
5-6-7-5
7-9-9-4
9-2-10-8
48-8-63-0
Mean
(MS);
10-4
6-5
8-6
10-0§
55-9
Time required for
completion
(months)
4
4
4
4
14-16
•One or two required.
t Average of low estimates-average of high estimates.
{Thousands of dollars.
§lt is anticipated that studies using rabbits would cost 15-20% more than those
using rats. The apparent discrepancy is attributed to the fact that laboratories
submitting the highest quotes for rats did not submit estimates for rabbits.
to pregnant females during the sensitive period of organogenesis. The foetuses are obtained
by Caesarean section prior to the expected parturition date and are observed for evidence
of abnormalities. Several species may be used, including the rat, mouse, hamster, rabbit or
monkey. The cost data obtained for these studies in rats were $7373-$13,467 for the average
range, with a mean of $10,420. Studies in mice and hamsters are in the same price range.
Generally, rabbits are a little more expensive, and studies in monkeys are 5-10 times more
costly. Tests in all species except monkeys require about 4 months to complete, but at least
1 year is necessary in the case of monkeys.
The long-term toxicity studies arc conducted primarily for evaluation of carcinogenic
potential (Table 6). Test animals may be the rat, hamster or mouse, although the dog has
Table 6. Cost/time estimates for chronic feeding studies
No. of .
Species* quotes
Dog (2 yr) 12
Rat(2yr) 13
Hamster (2 yr) 7
Mouse (18 months) 8
Total for two
species (rat and dog) .
Cost
Average rangef
' (MS);
75-9-89-2
68-5-77-1
45-8-52-3
45-2-49-3
. . 144-4-166-3
Mean
(MS);
82-6
72-8
49-0
47-2-
155-3
Time required for
completion
(months)
29-30
28-29
28-29
27-28
28-30§
*Two required.
fAverage of low estimates-average of high estimates.
{Thousands of dollars.
§If run concurrently.
54
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1104 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
been used extensively in the past. Tests in two rodent species are now being suggested,
lasting 2 years except for those in mice, which run for 18 months.
Residue data and subacute studies may indicate the necessity for chronic studies also
on a metabolite which would double the cost. As currently conducted, these studies follow
the same general design as the 90-day studies. More animals are started on the test, at least
25/sex/dose and frequently 40-100/sex/dose, to assure sufficient numbers at termination.
Special attention is given to tumour formation, but otherwise clinical and pathological
evaluations are similar to those in the subacute studies.
At present, there is not a firm requirement for mutagenicity studies, although they are
proposed and are likely to become part of the testing programme (Table 7). The relevance
Table 7. Cost/time estimates for mutagenicity studies
Test
Host-mediated assay
Dominant lethal
Cytogenetic studies
No. of
quotes
7
9
7
Total .
Mean cost
(MS)*
1-7
8-1
7-3
.. 17-lf
Time required for
completion
(months)
2-3
4
4
4J
*Thousands of dollars.
tAverage range (average of low estimates-average of high estimates), MS 15-0-
19-4.
JIf run concurrently.
of the results for assessing human hazard, however, is still open to question. Three studies
are recommended: ,
Host-mediated assay. This system involves injection of bacterial organisms into the
peritoneal cavity of a mammalian host, followed by administration of the test material
to the host by a different route. The bacteria arc then recovered and examined for the
induction of mutants. This study ranges in average cost between $1468 and $1968,
mean $1718. Approximately 2-3 months are required.
Dominant lethal test. This involves mating treated male rats, usually each week for 7
weeks after treatment, with untreated females and observing mortality in utero. The
average range of costs from nine laboratories was $7330-$9000 with a mean of $8170.
The time required is approximately 4 months.
Cytogenetic studies. In vivo cytogenetic studies consist of examination of the chromo-
somes of treated animals and sometimes of exposed human beings for aberrations.
These studies take about 4 months and cost on average from $6236 to $8380, mean
$7310.
The total cost of the toxicological evaluation of a pesticide, then, would range from
$306,800 to $375,480 at a minimum, with a mean of $349,800 (Table 8). The studies would
take a minimum of 4 years, and more probably 6 years, to complete, depending upon how
the programme was scheduled. The addition of various other studies, such as extra sub-
acute investigations, human studies, mutagenicity studies, teratology on another species
and chronic studies on a metabolite would bring the total to about $625,600. These are very
likely additions and are the rule rather than the special-case exception. This is by no mea'ns
55
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TOXICOLOGY: COST/TIME 1105
Table 8. Summary of cost/time estimates for toxicity testing
Mean cost
Test (MS)*
Minimum requirements
Acute 18-4."
Subacute 100-5
Metabolism, rat and dog 29-8
Reproduction, rat 35-4
Teratology, rat 10-4
Chronic feeding, rat and dog 155-3
Total. . . 349-8f
Additional tests
Subacute 13-2
Teratology, second species 10-0
Human metabolism 21-1
Mutagenesis 17-2
Human toxicology 50-0
Chronic feeding, metabolite 155-3
Total.. . 275-8
•Thousands of dollars.
tMinimum time required for completion, 48 months.
the upper limit, either. In some instances chronic inhalation studies may be required for
evaluation of carcinogenicity and these would cost in the range of $80,000-$125,000,
bringing the total to almost $750,000.
The interest which the money expended for these studies would draw constitutes an
additional substantial cost. Also not considered are the additional costs of administration,
consultation and liaison, which may add 20% or more to the cost of a study.
This programme for obtaining acceptable toxicity data on pesticides involves large
amounts of time and money. .Moreover, the impact of the results is considerable, in that
safety factors and tolerances are derived from them, and theoretically the health and well-
being of people and their environment may depend upon the proper conduct of the tests
and evaluation of the data. It is therefore appropriate to examine some of the aspects of
these studies, especially regarding design and interpretation. A detailed critical evaluation
of all the test methods would fill volumes, but a few points have been the subject of much
discussion and bear summarizing here.
First, a brief mention is in order of how the actual test systems used have become estab-
lished. Although protection of the public's health, food supply and environment from
harmful amounts of pesticides has been provided for by legislation, the specific test methods
employed in toxicology to provide data for safety evaluation have evolved through the
years according to "the state of the art". Usually the test methods, both in their general
approach and specific details, have been adopted as a result of recommendations and
guidelines issued by a regulatory agency, either USDA, the FDA, or currently the EPA.
They are not legislation or "law", but although allegedly this is not the intent, they become
de facto minimum standards, and when another experimental approach seems equally or
more appropriate, it must be undertaken in addition to, and not instead of, the official
decree. Because of this shift from recommendation to required standard, newly proposed
official guidelines should be, and usually are, challenged if considered inordinately restrictive,
56
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1106 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
scientifically unsound or impractical, by experienced members of the lexicological com-
munity. Unfortunately, the track record of such challenges has been very poor.
Several factors in the design and conduct of repeated exposure experiments have prompted
considerable discussion. First there is the selection of species of animal to be used. There
are a few prerequisites regarding this. There are practical considerations, such as size,
availability, ease of maintenance and handling and short lifespan. Good background
information regarding normal clinical values, mortality, tumour incidence and other
characteristics is absolutely essential. The ideal choice is the species that metabolizes a
given chemical in the same manner as does man.
The scheme for determining this most appropriate test species has been described by
various groups (Foodand Cosmetics Toxicology, 1969; Food Protection Committee, 1970;
Frazer, 1970; Frazer & Sharratt, 1969). Generally, it involves preliminary subacute studies
in several species, from which data regarding both toxicity and metabolism can be obtained.
Then, if not contraindicated by high toxicity, limited tests in man are undertaken for
determination of metabolites in blood, urine and faeces. At this point it may be possible to
make a reasonably good choice of species for longer-term studies. This is an extreme
oversimplification of the problem for most cases, however. Numerous species may be tested,
still without good correlative data. In the case of coumarin, ten species were examined before
one was found that was similar to man in its metabolism of this compound (Food and
Cosmetics Toxicology, 1970; Kaighen & Williams, 1969; Shilling, Crampton & Longland,
1969). If the species were an exotic one, difficult to handle or without adequate background
information, the disadvantages could well override the advantage of an apparent metabolic
similarity. The value of the information gained must be balanced against opposing factors.
The species commonly used in repeated toxicity testing have various advantages and
drawbacks. The rat is the most widely used, for both its practicality and physiology. There
is general agreement as to the value of studies in rats (Edson, Noakes & Sanderson, 1968;
Frazer, 1969). The dog is |the non-rodent species of choice for subacute testing, but its long
lifespan renders it inappropriate for carcinogenic evaluations. This raises the point of the
rationale of 2-year feeding studies in dogs. These studies are intended for evaluation of
carcinogenicity and extend for the lifetime of other species. Furthermore, Weil & Mc-
Collister (1963), in comparisons of 21 long-term rat and dog studies, showed that dogs were
no more sensitive than rats regarding other indications of toxicity. Therefore, it would seem
logical to conduct studies of shorter duration in dogs since it would be much less expensive
and just as valuable, as long as attempts are not made to ascertain carcinogenicity. The dog
is susceptible to bladder carcinomas induced by certain aromatic amines, where other species
have proved refractory (FDA Advisory Committee on Protocols for Safety Evaluation,
1971). In these cases, the 7-ye'ar or more experimental period is warranted. There are
indications that the hamster may prove to be a satisfactory substitute.
Mice are used in chronic studies, especially those for the evaluation of carcinogenic
potential. They are, however, very susceptible to a wide variety of spontaneous and induced
tumours and their suitability for a reasonable carcinogenic investigation has been questioned
(Grasso & Crampton, 1972).
Monkeys have received considerable attention as test animals. There is no evidence,
however, that they are closer to man with regard to metabolic patterns than are rats or dogs
(Committee on Problems of Drug Safety, 1969; Frazer, 1969; Noel, 1970). In fact, being
herbivores, evolutionary adaptive mechanisms have no doubt left them quite unsuitable
for evaluating the toxicity of many agents.
57
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TOXICOLOGY: COST/TIME 1107
Various other animals have been used in special cases. The use of neonatal mice and ham-
sters has been in vogue in the last few years for carcinogenicity testing and has met with
criticism, as the interpretation of results is most difficult (Food and Cosmetics Toxicology,
1968; Task Force on Research Planning in Environmental Health Science, 1970).
Earlier in this paper, mention svas made of the standard clinical tests that are determined
in subacute and chronic feeding studies. A few of these will be discussed in more detail at
this point. Urine analyses are routinely conducted, although results are almost always
normal, even in cases of morphological renal damage. Blood urea nitrogen, the most
common determination for evaluation of renal function, is likewise very insensitive, normal
levels being found even with severe renal damage (Sharratt, 1970). Measurement of brom-
sulphthalein (BSP) dye retention is a liver-function test routinely conducted in dogs.
However, up to 50% of the functional capacity of the liver must be impaired before the dye
test will indicate the damage (Cornish, 1971). There seems to be little scientific basis, then,
for routinely conducting these tests. Rowe, Wolf, Weil & Smyth (1959) and Weil & Mc-
Collister (1963) compiled and compared data from a number of short- and long-term
feeding studies and found, among other things, that the most efficient criteria for defining
the lowest dosage level that produces an effect are body-weight gain, liver and kidney
weights and pathological alterations in the liver and kidney. This is not to suggest that these
should be the only criteria examined, but that certain clinical studies should be included
only when indicated and not run routinely according to some "recipe". Furthermore, even
if omission of these tests resulted in missing a change (however unlikely), the effect would be
detected by other, more sensitive criteria, so there would be no loss of information.
Weil & McCollister (1963) also found that results of 90-day studies are excellent predictors
of the outcome of 2-year trials. Other investigators share the belief that studies of a few
months duration will reveal the nature of important toxic effects, except for carcinogenicity
(Paget, 1963 & 1968). Thus long-term studies could truly concentrate on the evaluation of
carcinogenic potential, rather than be cluttered up with meaningless and repetitious data.
The selection of dosage levels administered in repeated dose studies is an important
decision. Doses for subacute studies may be estimated from LD50 data, but this is often not
very successful. Weil, Woodside, Bernard & Carpenter (1969) have shown short-term
(7-day) tests to be much more efficient in predicting optimal dose ranges for longer term
studies. In well-designed toxicological investigations, a range of doses is administered so
that dose-related effects will be evident and a safe threshold dose can be calculated. The
establishment of this dose-response relationship is "fundamental to all toxicological
endeavour" (Golberg, 1971).
There are those in the field who advocate the administration of massive doses, presum-
ably in an attempt to maximize the likelihood of toxic manifestations. One wonders if this
might be partly due to an aversion to. negative data. Striking results attract much more
attention, both from certain scientific publications and from the lay media. However, the
effects fronr excessive doses may simply be evidence of abnormal metabolism as a result
of overloading the system, and are not necessarily reflections of inherent toxicity or charac-
teristic of changes occurring at lower doses (Food Protection Committee, 1959). It is likely
in many instances that normal metabolism could handle the chemical in more reasonable
quantities (idem, 1970). Data obtained from animals given massive, unrealistic doses are
especially inappropriate for assessing hazard in the light of the current emphasis on the
effects of exposure to very low levels of pesticides over extended periods, perhaps a lifetime.
The practice of administering massive doses really becomes a serious problem when a
58
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1108 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
zero tolerance is applied on the basis of the results. This is true in the case of suspected
carcinogens, as provided by the Delaney Clause, which states that no level of an additive
is safe if it is found to induce cancer in man or animals by appropriate test procedures.
This sounds like a noble pronouncement, until one realizes that the qualification in the
clause relating to appropriateness is often ignored in designing experiments and interpreting
results. A definition of appropriateness is definitely in order, so that our food supply will
not be jeopardized by results from poorly designed, irrelevant toxicity studies.
Although not included in the Delaney Clause, the concept of "zero tolerance" has been
extended by some people to cover agents that have been shown to be teratogens and muta-
gens, regardless of dosage, in animal studies. Various factors which may influence foetal
development are notorious. Golberg (1971) has summarized these, which include transport
of mice by air on days 12 and 13 of pregnancy, fasting at a critical stage or a diet of
raisins for one day, along with other stress factors. Interpretation of results from muta-
genicity studies is in an extremely nai've stage, with really no understanding of how the find-
ings might apply to man. This is not to suggest that these new test systems be abandoned
or ignored. However, before high-impact decisions are reached on the basis of the results,
more progress should be made in evaluating the significance of findings, including con-
sideration of dose-response relationships.
Since many of the problems in toxicity studies arise in the interpretation of data rather
than in methodology, a few more words on this subject are in order here. Reverence is often
made to "the level of no effect" defined by toxicity investigations, implying that thousands
of dollars and sometimes years of effort have been reduced to a single number. Indeed the
significance of this expression should not be minimized; not only is it of practical importance
in that it is used in calculations of safety factors and tolerances, but behind it lies the primary
concept in toxicology—that there is a safe level of virtually any material, below which
"there is practical certainty that injury will not result. . ."; this is safety, as defined by the
Food Protection Committee (1959). Arriving at a no-effect level for a given compound
requires judgement based o^ broad experience in toxicology. The concept of the term is a
dynamic one, which has to be re-evaluated with each new, more sensitive test system.
One of the major interpretative problems in toxicology is that of evaluating the significance
of subtle changes detected by various criteria. How these are interpreted has a definite
influence on the final no-effect level defined. One of the most obvious examples of this kind
of change is that of increased liver weights. Chapters have been written regarding the sig-
nificance of this, and there is still some lack of agreement, although the general consensus
seems to be that, in the absence of functional and morphological changes in the liver, this
effect is without toxicological significance.
There are other examples of interpretative problems, but space does not permit elabora-
tion. In general though, dilemmas still exist in distinguishing between "response and injury;
no harmful effect and no detectable effect; adaptation and damage; permanent and transient
damage; an acceptable and an unacceptable positive finding . . ." (Frazer, 1969).
Along with the enormous task of solving some of the interpretative problems just men-
tioned, there are other situations that warrant high-priority attention. More consideration
should be given to naturally occurring toxins; their contribution to the exposure level of
carcinogens, mutagens and teratogens should be explored. The benefits of pesticides should
be weighed very carefully against the risk involved. The production of food without suitable
pesticides is a frightening prospect and hasty decisions should be avoided except in a case
of a real public health hazard.
59
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TOXICOLOGY: COST/TIME 1109
Conclusions
It is very clear from this information that obtaining toxicity data on pesticides for
evaluation of safety is an expensive, time-consuming procedure. Moreover, the impact of
the results is considerable in that tolerances are derived from them and theoretically the
health and well-being of people and their environment may depend upon the proper
conduct of the test and evaluation of the data.
Often overlooked are the conservative judgements made in establishing a safe level for a
pesticide. After a "no-effect" level of a pesticide for laboratory animals is established, a
safety factor of 100 is frequently used in extrapolating to a safe level of intake for man,
a level commonly designated as the ADI, the Acceptable Daily Intake. A tolerance level
for a given pesticide established by regulation takes into consideration not only the pro-
jected safe level, but also the actual residue levels that result from the prescribed use in
various commodities and the consumption patterns of the individual commodities.
Although some of the tests in question may be scientifically valid in themselves, they are
inappropriately designed and/or interpreted to enhance the establishment of safety. Many
procedures in toxicological testing are not only costly but redundant. Even more disturbing
is the fact that many of the redundant procedures are less sensitive than others for detecting
an untoward effect. These low-yield, high-cost procedures are performed to satisfy whims
rather than to provide data for safety evaluation.
Currently, these policies are at the expense of the pesticide manufacturers and therefore
ultimately the consumer. Hopefully, they will not render the pesticide business so pro-
hibitively expensive and the business risk so great that the food supply of the nation and
the world will suffer.
REFERENCES
Committee on Problems of Drug Safety (1969).'Application of metabolic data to the evaluation of drugs.
A Report prepared by the Committee on Problems of Drug Safety of the Drug Research Board, National
Academy of Sciences—National Research Council. Clin. P/iarmac. Ther. 10, 607.
Cornish, H. H. (1971). Problems posed by observations of serum enzyme changes in toxicology. CRC
Critical Reviews in Toxicology 1, 1.
Edson, E. F., Noakes, Diana N. & Sanderson, D. M. (1968). Pesticides. In Modern Trends in Toxicology, 1.
Edited by E. Boyland and R. Goulding, p. 267. Butterworths, London.
FDA Advisory Committee on Protocols for Safety Evaluation (1971). Panel on carcinogenesis report on
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Food and Cosmetics Toxicology (1968). Carcinogen testing in the newborn mouse, ibid 6, 655.
Food and Cosmetics Toxicology (1969). From mice to men. ibid 7, 675.
Food and Cosmetics Toxicology (1970). A fresh look at acute toxicity. ibid 8, 214.
Food Protection Committee (1959). Principles and Procedures for Evaluating the Safety of Food Additives.
National Academy of Sciences-National Research Council Publication 750, Washington, D.C.
Food Protection Committee (1970),Evaluating the Safety of Food Chemicals. National Academy of Sciences-
National Research Council, Washington, D.C.
Frazer, A. C. (1969). Discussion. In The Use of Animals in Toxicological Studies. Universities Federation for
Animal Welfare Symposium, p. 38. UFAW, Potters Bar, Herts.
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Metabolic Aspects of Food Safety. Edited by F. J. C. Roc. p. 1. Blackwell Scientific Publications, Oxford.
Frazer, A. C. & Sharratt, M. (1969). The value and limitations of animal studies in the prediction of effects.
In The Use of Animals in Toxicological Studies. Universities Federation for Animal Welfare Symposium,
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Golberg, L. (1971). Trace chemical contaminants in food: Potential for harm. Fd Cosmet. Toxicol. 9, 65.
Grasso, P. & Crampton, R. F. (1972). The value of the mouse in carcinogenicity testing. Fd Cosmet. Toxicol.
10,418.
Kaighen, M. & Williams, R. J. (1969). The metabolism of [3-'*C] coumarin. /. mednlpharm. Ckem. 3, 25.
McCollister, D. D. (1965). Safe Tolerances for Food and Milk. Proceedings of the Idaho Annual Health
Conference, Sun Valley, Idaho, p. 28.
F.C.T. 11/6—M ....
QU
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1110 P. J. GEHRING, V. K. ROWE and SUSAN B. MCCOLLISTER
Noel, P. R. B. (1970). The challenge of selecting the suitable species in toxicology: Non-rodents (particularly
the dog). Proceedings of the European Society for the Study of Drug Toxicity, Vol. XI: Excerpta Mcdica
Foundation, Amsterdam.
Paget, G. E. (1963). Standards for the laboratory evaluation of the toxicity of a drug. In Proceedings of the
European Society for the Study of Drug Toxicity. Vol. II. p. 7. International Congress Series no. 73.
Excerpta Medica Foundation, Amsterdam.
Paget, G. E. (1968). Drug safety. In Modern Trends in Toxicology, 1. Edited by E. Boyland and R. Goulding.
p. 231. Buttcrworlhs, London.
Rowe, V. K., Wolf, M. A., Weil, C. S. & Smyth, H. F., Jr. (1959). The lexicological basis of threshold limit
values: 2. Pathological and biochemical criteria. Am. ind. Hyg. Ass. J. 20, 346.
Sharratt, M. (1970). Renal function tests in laboratory animals. In Metabolic Aspects of Food Safety. Edited
by F. J. C. Roe. pp. 119 & 124. Blackwell Scientific Publications, Oxford.
Shilling, W. H., Crampton, R. F. & Longland, R. C. (1969). Metabolism of coumarin in man. Nature,Land.
221, 664.
Task Force on Research Planning in Environmental Health Science (1970). Carcinogenesis, Mutagenesis
and Teratogenesis in Man's Health and the Environment—Some Research Needs, pp. 153 & 157.
US Department of Agriculture (1968). The Regulation of Pesticides in the United States, p. 2. USDA,
Washington, D.C.
Weil, C. S. & McCollister, D. D. (1963). Safety evaluation of chemicals. Relationship between short- and
long-term feeding studies in designing an effective toxicity test. J. agric. Fd Chtm. 11, 486.
Weil, C. S., Woodside, M. D., Bernard, J. R. & Carpenter, C. P. (1969). Relationship between single-
peroral, one-week, and ninety-day rat feeding studies. Toxic, appl. Pliarmac. 14, 426.
61
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REPEATED DERMAL APPLICATION TO RABBITS
ANIMALS; Fifty albino rabbits (New Zealand white), two to
three kilograms in body weight, will be placed on experiment
as follows:
Group Number per Group
I 2 males, 2 females
II 5 males, 5 females
5 males, 5 females
III 5 males, 5 females
5 males, 5 females
Skin Condition
Intact
Intact
Abraded
Intact
Abraded
Dosage
Control
High
Low
COMPOUND ADMINISTRATION; Applied dermally to the prepared area
of the back of each treated rabbit, once daily, five days
per week for a period of three weeks (15 applications).
Appropriate amounts of the formulation vehicle will be
applied to the backs of the control rabbits in the same
manner. After application, the exposed area will be
covered with loosely fitting gauze to minimize the possi-
bility of compound ingestion. All residual material will
be removed prior to each daily application.
DURATION; Three weeks.
CLINICAL OBSERVATIONS; General observations daily; body weights
and physical condition weekly.
HEMATOLOGY; Hemograms consisting of hemoglobin, hematocrit, and
total and differential leucocyte counts to be conducted on
all animals initially and at termination.
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CHOLINESTERASE INHIBITION; Both RBC and plasma determinations
to be run on all animals initially and at termination.
NECROPSY; To be performed on any animal dying or at the point
of death and all survivors sacrificed at termination. All
important organs and tissues will be examined and sections
of liver, kidney, thyroid, gonads, adrenals, and appli-
cation sites will be taken and preserved in 10 per cent
formalin.
HISTOPATHOLOGY; The above named tissues will be examined in
respect of all control and high level animals. Histo-
pathological examination of tissues from low level animals
will be conducted if indicated.
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REPEATED ORAL ADMINISTRATION TO MALLARD DUCKS
PROCEDURE; Approximately 200 five-day-old mallard ducks
.will be used to conduct this study. A sufficient
number of graded dose levels of the test materials
(technical and formulation) will be administered in
the diet to groups of 10 birds for seven days. The
• birds will be held on basal ration for an additional
three days, then sacrificed and representative birds
examined for gross pathological changes. Untreated
control groups will be maintained in a similar
manner.
Using such parameters as mortality, gross pathological
changes, signs of intoxication, food consumption, and
body weight changes, the possible hazard of the test
material will be evaluated.
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ACUTE ADMINISTRATION TO SUNFISH
PROCEDURE; Bluegill sunfish will be acclimated for a mini-
mum of seven days. During this acclimation period,
the fish will be fed Purina Trout Chow daily. For
each test material, a population of approximately
110 fish of uniform size (approximately 1.5 grams in
weight and 4.0 cm in length) will be selected and
fasted for 72 hours.
The water in which the fish are held during the period
will be deionized tap water, reconstituted by adding
CaS04 (30 mg/1), MgSO4 (30 mg/1), NaHCO3 (48 mg/1),
and KC1 (3 mg/1). The water will be aerated prior to
the test period.
Each test material will be dissolved in a suitable
solvent and aliquots will be pipetted into test jars
containing 15 liters of reconstituted water to attain
desired levels of the test material in terms of parts
per million (ppm).
A sufficient number of groups of 10-20 fish per group
will be placed in the jars and exposed to the graded
levels of the test material for a period of 96 hours.
Observations will be made at regular intervals for signs
of intoxication and mortality. An LC5Q for each test
material will be determined at 48 and 96 hours.
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TERATOLOGICAL STUDY IN RATS
ANIMALS; Sixty pregnant female albino rats (day of mating
established) will be placed on experiment as follows:
Group Number per Group Dosage Level*
I 20 Control
II 20 High
III 20 Low
* Actual dose levels to be established
COMPOUND ADMINISTRATION; The test material will be admin-
istered in the diet to the Group II and III rats from
day 6 to day 15 of the gestation period.
PROCEDURE; All females will be weighed on days 0, 6, 15,
and 20 of gestation and food consumption recorded
during the dosing period.
The numbers of fetuses, their placement in the uterine
horns, correlation with the numbers of corpora lutea,
live and dead fetuses, and resorptions will be deter-
mined.
All fetuses will be weighed and examined closely for
externally observable signs of abnormality. One-third
of the fetuses will be examined for visceral abnormal-
ities and two-thirds cleared, stained, and examined for
skeletal abnormalities.
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The data are to be reported in the following format:
Observations Made Results
Dose Levels
Average Body Weight of Dams(g)
Mating Day 1
Gestation Day 0
Gestation Day 6
Gestation Day 15
Gestation Day 20
Matings Resulting in Conceptions
Average Fetal Weight(g)
Average Litter Weight(g)
Total Implantation Sites
Average Implantation Sites/Litter
Implantation Sites Left Horn
Implantation Sites Right Horn
Total Viable Fetuses
Total Dead Fetuses
Total Resorption Sites
Per Cent Resorptions
Gross Abnormalities
Stained Skeletal Abnormalities
Bouin's Section Abnormalities
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TERATOLOGICAL STUDY IN RABBITS
ANIMALS; Female albino rabbits of breeding age will be
acclimated to laboratory conditions and placed on
experiment as follows:
Pregnant Females
Group Per Group Dosage Level
I 10 Control
II 10 High
III 10 Low
A sufficient number of untreated adult male rabbits
will be used for mating, or alternatively artificial
insemination will be used.
COMPOUND ADMINISTRATION; The test material will be admin-
istered in the diet to the Group II and III animals
from day 6 to day 18 of gestation.
OBSERVATIONS; All females will be weighed on days 0, 6,
12, 18, and 30 of gestation. Food consumption will be
recorded during the dosing period.
On day 30 of the gestation period, all pregnant females
will be sacrificed and the fetuses delivered by Caesarean
section. The ovaries and uterus of each parent female
will be examined for corpora lutea, implantation sites,
resorptions, and other evidence of abnormal reproductive
processes.
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The numbers of viable and non-viable fetuses will be
recorded. All surviving offspring from each litter will
be placed in an incubator by litter groups for observa-
tion of 24-hour survival. All surviving fetuses will be
sacrificed and these, along with all non-survivors, will
be examined closely for externally observable signs of
abnormality. The fetuses will then be subjected to
detailed necropsy and examined for any evidence of vis-
ceral abnormality, then cleared, stained, and examined
for evidence of skeletal abnormalities.
The data are to be reported in the following format:
Observations Made Results
Dose Level
Maternal Mortality
Average Body Weight of Dams(kg)
Day 0
Day 6 .
Day 12
Day 18
Day 30
Percent Conception
Percent Alive at C-section
Percent Resorption
Percent Dead
Average Litter Size
Average Viable Litter Weight (g)
Average Pup Weight (g)
Incidence of Abnormalities (%)
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ACUTE INHALATION EXPOSURE TO RATS
PROCEDURE; Groups of 10 young adult albino rats each will
be placed in a hynamic exposure chamber and exposed
to the test material as the formulation at different
levels in an effort to establish the approximate LCj-n
of the test material. The amount of exposure will be
varied by chamber concentration of the test material
or time of chamber exposure of the animals.
Following each exposure, the animals will be removed
and the number of survivors, signs of respiratory in-
volvement, and eye irritation noted and recorded.
The survivors will be observed daily thereafter for
14 days or until death. At 14 days, all survivors
will be sacrificed.
Gross necropsy will be performed on all animals dying
as well as sacrificed survivors at termination.
Important organs will be weighed and grossly examined,
Sections of major tissues will be preserved in 10%
formalin in the event histopathological observations
are indicated.
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SINGLE ORAL ADMINISTRATION TO RATS
PROCEDURE; Approximately 30 male and 30 female young adult
albino rats each w,
the test material.
albino rats each will be used to estimate the LD5Q of
Following administration of graded doses of the test
material, the animals will be observed closely for
pharmacologic and toxicologic signs throughout the day.
All rats will be closely observed, daily thereafter,
for a period of 14 days or until death for signs of
delayed toxicity. All rats that die during the obser-
vation period and representatives of survivors sacri-
ficed at termination will be subjected to gross necropsy,
Weights at 0, 4, 9, and 14 days will be recorded.
Calculations of the LD will be determined by the
method of H. J. Horn (Biometrics, 12,:311, 1956).
Alternate Method: Computer calculated Probit Analysis,
D. J. Finney (Cambridge University Press, 1952).
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SINGLE ORAL ADMINISTRATION TO RABBITS
PROCEDURE; Sixteen to twenty young adult rabbits (New
Zealand white) will be used to estimate the LD^Q of
the test material.
Following administration of graded doses of the test
materials, the animals will be observed closely for
pharmacologic and toxicologic signs throughout the
day. All rabbits will be observed closely, daily
thereafter, for a period of 14 days or until death for
signs of delayed toxicity. All rabbits that die during
the observation period and representatives of survivors
sacrificed at termination will be subjected to gross
necropsy. Weights at 0, 4, 9, and 14 days will be re-
corded.
Calculations of the LD5Q will be determined by the
method of H. J. Horn (Biometrics, 1^:311, 1956).
Alternate method: Computer calculated Probit Analysis,
D. J. Finney (Cambridge University Press, 1952).
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SINGLE ORAL ADMINISTRATION TO DOGS
PROCEDURE; Six to eight normal mongrel dogs will be used to
estimate the approximate lethal dose of the test material
when administered.
Following administration/ the animals will be observed
closely for pharmacologic and toxicologic signs through-
out the day. All dogs will be observed closely, daily
thereafter, for a period of 14 days or until death for
signs of delayed toxicity. All dogs that die during
the observation period will be subjected to gross
necropsy. Weights at weekly intervals, two weeks prior
to and two weeks following compound administration will
be recorded.
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REPEATED ORAL ADMINISTRATION TO RATS
FOR 13 WEEKS
ANIMALS; One hundred twenty albino rats obtained at weaning
from The Charles River Breeding Laboratories, Inc.,
housed individually in temperature-controlled quarters,
and acclimated to laboratory conditions for one week
will be placed on experiment as follows:
Group Number per Group Dosage Level*
I 15 males, 15 females Control
II 15 males, 15 females High
III 15 males, 15 females Intermediate
IV 15 males, 15 females Low
* Actual dose levels to be established
COMPOUND ADMINISTRATION; By incorporation in the diet by
mechanical mixer with levels adjusted to maintain a
relatively constant intake of test material in terms
of mg/kg/day.
DURATION: Thirteen weeks
CLINICAL OBSERVATIONS; General observations daily; body
weights, food consumption, and physical examination
weekly.
HEMATOLOGY; Hemograms consisting of hemoglobin, microhemato-
crit, coagulation time, thrombocyte counts, and total
and differential leucocyte counts will be determined on
5 males and 5 females each from Groups I and II at 0, 4,
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8 and 13 weeks. Lower levels will be examined when
indicated by results in Group II.
CLINICAL CHEMISTRY; Blood glucose, prothrombin time, and
serum glutamic pyruvic transaminase will be determined
on 5 males and 5 females each from Groups I and II at
0, 4, 8 and 13 weeks. Lower levels will be examined
when indicated by results in Group II.
NECROPSY; To be performed on any animal dying or at the
point of death and all survivors sacrificed at term-
ination.
The following organs will be weighed: heart, lung, liver,
kidneys, spleen, gonads, adrenal, thyroid, prostate or
uterus, and pituitary. Portions of these organs plus
portions of the following tissues will be grossly exam-
ined and preserved in 10 percent formalin: duodenum,
intercostal muscle, urinary bladder, pancreas, mesenteric
lymph node, mammae, bone marrow, stomach, and brain. In
addition, eyes will be taken, grossly observed, and pre-
served in Zenker's fixative.
HISTOPATHOLOGY: The above named tissues (approximately 18 per
rat) will be examined from any animal dying when the tissues
are in good condition and from 5 males and 5 females each
from Groups I and II sacrificed at termination. In addi-
tion, eight selected tissues will be examined from 5 males
and 5 females each, from Groups III, IV and V sacrificed
at termination. Additional tissues will be examined from
lower levels if indicated by results from Group II.
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REPEATED ORAL ADMINISTRATION TO DOGS
(OR MONKEYS) FOR 13 WEEKS
ANIMALS: Twenty-four purebred beagle dogs, six to eight
months of age, individually housed in temperature-
controlled quarters, properly treated for intestinal
parasites, immunized against rabies, distemper, hepa-
titis, and leptospirosis, and acclimated to laboratory
conditions for three weeks will be placed on experiment
as follows:
Group Number per Group Dosage Level*
I 3 males, 3 females Control
II 3 males, 3 females High
III 3 males, 3 females Intermediate
IV 3 males, 3 females Low
* Actual dose levels to be established
COMPOUND ADMINISTRATION; By gelatin capsule, once daily, seven
days per week, with empty gelatin capsules to controls.
DURATION; Thirteen weeks
CLINICAL OBSERVATIONS; General, observations consisting of
behavior, food intake, stool consistency, etc., daily;
body weights and detailed physical examination weekly.
In addition, heart rate and blood pressure will be deter-
mined and electrocardiograms and ophthalmic examinations
will be conducted on all animals initially and at 4, 8
and 13 weeks.
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HEMATOLOGY: Hemograms consisting of hemoglobin, hematocrit,
sedimentation rate, coagulation time, thrombocyte counts,
and total and differential leucocyte counts will be
determined on all animals twice initially and at 4, 8
and 13 weeks.
CLINICAL CHEMISTRY; Blood urea nitrogen, serum alkaline
phosphatase, blood glucose, prothrombin time, serum
glutamic pyruvic transaminase, and serum glutamic
oxalacetic transaminase will be determined on all
animals twice initially and at 4, 8 and 13 weeks.
URINALYSES; Qualitative urinalyses consisting of general
appearance, pH, specific gravity, albumin, glucose,
and microscopic examination of urinary sediment will
be determined on cage-collected samples from all animals
initially and at 4, 8 and 13 weeks.
NECROPSY; To be performed on any animal dying or at the
point of death and all survivors sacrificed at term-
ination.
The following organs will-be weighed: heart, lungs,
liver, kidneys, spleen, thyroid, adrenal, prostate or
uterus, gonads, pituitary, and brain. Portions of
these organs plus portions of the following tissues
will be grossly examined and preserved in 10 per cent
formalin: nerve, esophagus, duodenum, jejunum, ileum,
colon, cecum, stomach, pancreas, parotid salivary gland,
thymus, trachea, gall bladder, intercostal muscle,
urinary bladder, mesenteric lymph node, femoral bone
77
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marrow, spinal cord, mammae, and abdominal skin. In
addition, -eyes will be taken, grossly observed, and
preserved in Zenker's fixative.
HISTOPATHQLOGY; The above named tissues (approximately
32 per animal) will be examined from any animal dying
when the tissues are in good condition and from all
control and high level animals. In addition, 13 selected
tissues will be examined for each of the remaining animals,
Additional tissues will be examined from lower levels if
indicated by results from Group II.
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THREE-GENERATION REPRODUCTION STUDY IN THE RAT
ANIMALS; One hundred twenty albino rats, obtained at weaning
from The Charles River Breeding Laboratories, Inc., will
be acclimated to laboratory conditions for one week and
placed on experiment as follows:
Group Number per Group Dosage Level*
I 20 males, 20 females Control
II 20 males, 20 females High
III 20 males, 20 females Low
* Actual dose levels to be established
COMPOUND ADMINISTRATION; By incorporation in the diet. The
experimental material will be administered at appropriate
levels of concentration to all treated animals and their
surviving offspring beginning one week after weaning.
PROCEDURE:
1. When the above animals (FQ generation) are 100 days of
age, they will be paired to mate by placing a female
in a corresponding male's cage for a period of 10 days.
2. All offspring from this mating (F,a generation) will be
taken as soon after birth as possible, weighed, and
examined for externally observable signs of abnormality.
One-half of each litter of the new born will be preserved
in Bouin's solution and examined for visceral abnormalities
an one-half will be cleared, stained, and examined for
skeletal abnormalities. Stillborn animals will be simi-
larly examined.
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3. Immediately following the birth of the F,a generation
animals, the FQ females will be mated, this time
with a different male, producing the F,b generation.
Each litter will be reduced to 10 pups on day 5 and
all offspring will be weighed and examined on days 1,
5, and 21. The number of dead pups at birth, days 1-5,
and 6-21 will be recorded. Following the weaning of
these offspring, the parent animals will be sacrificed
and subjected to gross necropsy. Gonads will be weighed.
4. One or two animals of each sex from each F,b generation
litter will then be selected to give 20 males and 20 fe-
males per dosage level and the remaining weanlings
sacrificed.
5. These animals will be mated as before, producing an F-a
generation. The offspring will be weighed and observed
as described under Item 3 above and sacrificed at wean-
ing.
6. Ten days after the sacrifice of the F,,a weanlings, the
F,b females will be remated, this time with a different
male, producing the F2b generation. These offspring
will be weighed and observed,.and the parent (F,b)
animals sacrificed and necropsied, as described under
Item 3 above.
7. One or two animals of each sex from each F~b generation
litter will then be selected to give 20 males and 20 fe-
males per dosage level and the remaining weanlings
sacrificed.
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8. These animals will be mated as before, producing an F3a
generation. The offspring will be weighed and observed
as described under Item 3 above and sacrificed at weaning.
9. Ten days after the sacrifice of the F~a weanlings, the
Fpb females will be remated, this time with a different
male, producing an F.,b generation. These offspring will
be weighed and observed, and the parent (F~b) animals
sacrificed and necropsied, as described under Item 3
above.
10. At this time all F3b weanlings will be sacrificed and
subjected to gross necropsy. Heart, liver, and kidneys
from two pups per sex per litter will be weighed. Portions
of these plus portions of spleen, adrenals, thyroid,
gonads, and bone marrow from all F.^b animals will be
taken and preserved in 10 per cent formalin.
11. The above named tissues will be examined histopatho-
logically from one male and one female per each F.,b
generation litter; tissues will be examined from more
animals, if indicated.
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TEST FOR EYE IRRITANTS
Six albino rabbits are used for each test substance. Animal
facilities for such procedures shall be so designed and main-
tained as to exclude sawdust, wood chips, or other extraneous
materials that might produce eye irritation. Both eyes of
each animal in the test group shall be examined before testing,
and only those animals without eye defects or irritation shall
be used. The animal is held firmly but gently until quiet.
The test material is placed in one eye of each animal by gently
pulling the lower lid away from the eyeball to form a cup into
which the test substance is dropped. The lids are then gently
held together for one second.-and the animal is released. The
other eye, remaining untreated, serves as a control. For test-
ing liquids, 0.1 milliliter is used. For solids or pastes,
100 milligrams of the test substance is used, except that for
substances in flake, granule, powder, or other particulate form
the amount that has a volume of 0.1 milliliter (after compacting
as much as possible without crushing or altering the individual
particles, such as by tapping the measuring container) shall be
used whenever this volume weighs less than 100 milligrams. In
such a case, the weight of the 0.1 milliliter test dose should
be recorded. The eyes are not washed following instillation
of test material except as noted below.
The eyes are examined and the grade of ocular reaction is re-
corded at 24, 48 and 72 hours. Reading of reactions is facil-
itated by use of a binocular loupe, hand slit-lamp, or other
expert means. After the recording of observations at 24 hours,
any or all eyes may be further examined after applying fluor-
escein. For this optional test, one drop of fluorescein sodium
82
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ophthalmic solution USP or equivalent is dropped directly on
the cornea. After flushing out the excess fluorescein with
sodium chloride solution USP or equivalent, injured areas of
the cornea appear yellow; this is best visualized in a darkened
room under ultraviolet illumination. Any or all eyes may be
washed with sodium chloride solution USP or equivalent after
the 24-hour reading.
An animal shall be considered as exhibiting a positive reac-
tion if the test substance produces at any of the readings
ulceration of the cornea (other than a fine stippling), or
opacity of the cornea (other than a slight dulling of the
normal luster), or inflammation of the iris (other than a
slight deepening of the folds (or rugae) or a slight circum-
corneal injection of the blood vessels), or if such substance
produces in the conjunctivae (excluding the cornea and iris)
an obvious swelling with partial eversion of the lids of a
diffuse crimson-red with individual vessels not easily dis-
cernible.
The test shall be considered positive if four or more of the
animals in the test group exhibit a positive reaction. If
only one animal exhibits a positive reaction, the test shall
be regarded as negative. If two or three animals exhibit a
positive reaction, the test is repeated using a different
group of six animals. The second test shall be considered
positive if three or more of the animals exhibit a positive
reaction. If only one or two animals in the second test ex-
hibit a positive reaction, the test shall be repeated with a
different group of six animals. Should a third test be needed,
83
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the substance will be regarded as an irritant if any animal
exhibits a positive reaction.
To assist testing laboratories and other interested persons
in interpreting the results obtained when a substance is tested
in accordance with the method described in the first paragraph,
an "Illustrated Guide for Grading Eye Irritants by Hazardous
Substances" will be sold by the Superintendent of Documents,
Government Printing Office, Washington, D. C. The guide will
contain color plates depicting responses of varying intensity
to specific test solutions. The grade of response and the sub-
stance used to produce the response will be indicated.
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EVALUATION OF CARCINOGENIC POTENTIAL
IN RATS BY REPEATED ORAL ADMINISTRATION
ANIMALS; Four hundred eighty albino rats obtained at weaning
from The Charles River Breeding Laboratories, housed
individually in temperature-controlled quarters, and
acclimated to laboratory conditions for one week will be
placed on experiment as follows:
Group Number per Group Dosage Level*
I 60 males, 60 females Control
II 60 males, 60 females High
III 60 males, 60 females v Intermediate
IV 60 males, 60 females Low
* Actual dose levels to be established
DURATION; Approximately two years (life span of test animals) ,
COMPOUND ADMINISTRATION; By incorporation in the diet by
mechanical mixer with levels adjusted to maintain a rela-
tively constant intake of test material in terms of
mg/kg/day.
CLINICAL OBSERVATIONS; General observations daily; food con-
sumption weekly; body weights and detailed physical exam-
ination by biweekly intervals.
HEMATOLOGY; Hemograms consisting of hemoglobin, hematocrit,
sedimentation rate, coagulation time, thrombocyte counts,
and total and differential leucocyte counts will be deter-
mined on five males and five females each from Groups I
and II at 3, 6, 12, 18 and ^4 months.
85
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CLINICAL CHEMISTRY; Blood urea nitrogen, serum alkaline
phosphatase, blood glucose, prothrombin time, serum glu-
tamic pyruvic transaminase, and serum glutamic oxalacetic
transaminase (others as indicated) will be determined
on five males and five females each from Groups I and II
at 3, 6, 12, 18 and 24 months. In addition, plasma and
RBC cholinesterase will be determined initially and at
3, 6, 12, 18 and 24 months and brain cholinesterase at
termination if indicated.
URINALYSES; Qualitative urinalyses consisting of general
appearance, pH, specific gravity, albumin, glucose and
microscopic examination of urinary sediment will be
determined on cage-collected samples from five males and
five females each from Groups I and II at 3, 6, 12, 18
and 24 months.
NECROPSY; To be performed on any animal dying or at the point
of death. Portions of the following organs and tissues
will be grossly examined and preserved in 10 per cent
formalin: heart, lung, liver, kidneys, spleen, gonads,
adrenal, thyroid, prostate or uterus, pituitary, duodenum,
intercostal muscle, urinary bladder, pancreas, mesenteric
lymph node, bone marrow, stomach, brain, eyes and tissue
mass. A sample of brain tissue will be taken from five
males and five females each from Groups I and II and
frozen for cholinesterase determinations if indicated.
HISTOPATHOLOGY; Any suspected abnormal organ or tissues will
be examined with respect to all animals.
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