PB83-153916
SUBDIVISION F: PESTICIDE ASSESSMENT GUIDELINES
HAZARD EVALUATION - HUMAN AND DOMESTIC ANIMALS
Office of Pesticide Programs
Washington, DC
Qct 82
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NIIS
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PB83-153916
EPA 540/9-82-025
November 1982
PESTICIDE ASSESSMENT GUIDELINES
SUBDIVISION F
HAZARD EVALUATION:
HUMAN AND DOMESTIC ANIMALS
by
Office of Pesticide Programs
Guidelines Project Manager
Robert K. Hitch
Hazard Evaluation Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Office of Pesticide and Toxic Substances
Washington, D.C. 20460 •
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As a guideline for the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA), Subdivision F supports the data requirements of 40 CFR Part 158.
Subdivision F describes protocols for evaluating the toxicity of pesticides
to nonhuman organisms and for relating the results of these studies to human
safety evaulatlons. Comments from environmental groups, academia, and
industrial representatives were incorporated into the current draft.
Subdivision F constitutes one volume of a twelve-part FIFRA guidelines
series published by the National Technical Information Service.
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Foreword
Toxicity data related to human and domestic animal safety
evaluation are required to support pesticide registration under
the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).
Subdivision F describes study protocols which may be used to
develop these toxicity data. Public canment on Subdivision F was
accepted in a series of public meetings, the last of ich was
held in July 1982. Because of the importance of the issues raised
in the Subdivision F canments, Subdivision F was redraf ted with the
help of a review canmittee canprised of representatives fran
industry, environmental groups and academia. This guideline
refers to requirements established in 40 CFR Part 158 so that this
document can be read as a cauplete package and so that the protocols
may be explained in their proper context.
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SUBDIVISION F: HAZARD EVALUATION: HUMANS AND
DOME ST IC AN IMALS
Table of Contents
DISCUSSION PAGE
I. Organization and Phil ophy of
Subdivision F 1
II. The Standards for Acceptable Testing 3
III. Discussion of Individual Tests 12
A. Acute Oral, Dermal, and Inhalation Toxicity 12
B. Irritation and Sensitization Studies 13
C. Neurotoxicity Evaluations 13
D. Subthronic Studies 14
E. Chronic and Oncogenicity Studies 15
F. Teratogenicity and Reproduction Studies 16
G. Mutagenicity Studies 17
H. Special Testing 18
GUIDELINES
Series 80 Overview, Definition, and General Requires ents -
80—1 Overview 20
80—2 Definitions 20
80—3 General Provisions 21
80—4 Reporting of Data 28
80—5 Combined Testing 32
Series 81 Acute Toxicity and Irritation Studies
81—1 Acute Oral Toxicity Study 34
81—2 Acute Dermal Toxicity Study 39
81—3 Acute Inhalation Toxicity Study 44
81—4 Primary Eye Irritation Study 51
81—5 Primary Dermal Irritation Study 55
81—6 Dermal Sensitization Study 59
81—7 Acute Delayed Neueroboxicity of Organophosphorus
Subs tances 62
Series 82 Subchronic Testing
82—1 Subthronic Oral Toxicity (Rodent and Non-rodent):
90—Day Study 66
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Table of Contents (Continued)
GUIDELINES PAGE
82-2 Repeated Dose Dermal Toxicity: 21-Day Study 76
82—3 Subchronic Dermal Toxicity: 90—Day Study 83
82—4 Subthronic Inhalation Toxicity: 90—Day Study 92
82—5 Subchronic Neurotoxicity: 90—Day Study 103
Series 83 chronic and Long Term Studies
83—1 Chronic Toxicity Studies 107
83—2 Oncogenicity Study 117
83—3 Teratogenicity Study 125
83—4 Reproductive and Fertility Effects 130
83—5 Combined Chronic Toxicity/Oncogenicity Studies 137
Series 84 Mutagenicity
84—1 Purpose and General Recanmendations for Mutagenicity
- Testing 147
84—2 Mutagenicity Tests 148
Series 85 Special Studies
85—1 Metabolism Study 152
85—2 Danestic Animal Safety Testing 156
85—3 Dennal Absorption Studies of Pesticides [ Reserved]
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I. ORGANIZATION AND PHILOSOPHY OF SUBDIVISION F
[ NOTE; The following Discussion constitotes a reiteration of many parts
of the preamble to the Subpart F guidelines proposed on Aug. 22, 1978
(43 FR 37336). Much of that information, instruction, and explanation
apply to the current guidelines. Minor changes have been made to update
some of the preamble text to apply to the current guidelines. Most of
the preamble discussion material included in the 1978 preamble was
deleted in the current discussion.
General requirements and definitions which apply to many or all of
the sections in Subdvision F appear in the Overview and related sections,
in the § 80 series. For the rest of this subdivision, each kind of test
is described in a separate section of the guidelines. Similar tests are
grouped together in a series. The short term acute tests appear in the
§ 81 series; the subchronic tests appear in the § 82 series; and the long
term, chronic tests appear in the § 83 series. In addition, the § 84
series contains tests for evaluating mutagenic effects, and the § 85 series
contains special kinds of tests.)
A. General Information and Requirements .
1. Proposed rule, 40 CFR Part 158, specifies the kind of data and
information that must be suhnitted to EPA to support the registration
of each pesticide under the Federal Insecticide, Fungicide and
Rodenticide Act. The Agency intends to promulgate Part 158 as a final
rule during 1983. This subdivision provides detailed information
relating to the data requirements listed in 40 CFR Part 158 including the
conditions under which each data requirement is applicable, the standards
for acceptable testing, stated with as much specificity as the current
scientific disciplines can provide, and the information that should
be included in a test report.
2. Data requirements for manufacturing—use products . In the
Preamble to the 1978 proposed Guidelines, EPA asked for public c nment
on the question whether the data requirements of this subdivision
should be extended to manufacturing—use products. After serious
consideration of this issue, the Agency has concluded that extending
the data requirements to such pesticides is appropriate. The Agency
was influenced by the views of commenters on this issue who generally
favored a data suhnission requirement which makes the basic manufacturer
of an active ingredient responsible for providing nost of the environ-
mental fate data.
Therefore, a section of 40 CFR Part 158, entitled “Formulators’
Exemption” ( 158.50), requires a registrant of a manufacturing-use
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product to suhnit (or cite) any data pertaining to the safety of an
active ingredient in its product if the same data are required to
support the registration of an end—use product that could legally
be produced from the registrant’s manufacturing-use products. (An
end—use product is a pesticide product bearing label directions for
immediate end—use as a pesticide.) Section 158.50 also provides
that such data must be suhuitted by an applicant for registration
of end-use product, except that the producer of the end-use product
will generally not have to su1 nit or cite data pertaining to registered
products which the end—use producer purchases and uses to formulate
the end—use product. This decision reflects the Agency’s expectation
that manufacturing—use product registrants will be the major source
of registration data, and that end—use product formulators will, in
most cases, need to supply much less data. This decision is consistent
with the provisions of, and Congressional intent behind, § 3(c)(2)(D)
of FIFRA, which provides that:
No applicant for registration of a pesticide who proposes
to purchase a registered pesticide from another producer
in order to formulate such purchased pesticide into an
end—use product shall be required to--
(i) suk nit or cite data pertaining to the safety of
such purchased product; or
(ii) offer to pay reasonable compensation otherwise
required by § 3(c)(l)(D) of FIPRA for use of any such
data.
Implicit in § 3(c)(2)(D) is Congress’ expectation that
it would be the registrant of the manufacturing—use product who would
provide significant amounts of data pertaining to the safety of its
product. (See, e.g., Sen. Rep. No. 334, 95th Cong., 1st Sess.,
pp. 8-9.)
Moreover, if data requirements were imposed solely on registrants
of end—use products, § 3(c)(2)(D) might be read to prevent the Agency from
obtaining data on the grounds that the data pertain to the safety of a
purchased product.
B. “ Combined testing” Paragraphs .
Since EPA is concerned with efficient use of test animals, laboratory
facilities, and personnel resources, the Agency encourages applicants and
registrants to combine two or sore different assays into a single test.
In individual sections (See e.g. § 82—1 through —5 and § 83—1, —2 and
—5) assay systems are identified which could be combined, provided, of
ccurse, that the principles for both types of testing are met. In add!-
tion to the specific identified combinations, proposed § 80—5 states that
data required by this subdivision may be derived fr an tea t me tho dologi es
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which satisfy the principles for acceptable testing contained in two or
more different sections of this subdivision.
The Agency considers that published protocols which cover n re than
one assay system would be useful to some applicants and registrants.
Accordingly, as such protocols are developed, EPA will add them to the
lists of references in the appendices which appear at the ends of the test
sections.
C. “ Data reporting and evaluation” Paragraphs .
A paragraph describing the data reporting requirements follows the
t1 Principles” paragraph in each of the individual test sections. The
requirements in these paragraphs, together with the requirements in 80-4,
would prescribe the format and content of test data reports. Generally,
the test report shall include a summary and evaluation of the test results,
and specific detailed information to support the conclusions presented in
the summary.
The paragraphs specifying the data which shall be reported are quite
detailed and ask for considerable basic information. This information is
necessary, however, to permit Agency staff to make their own independent.
analysis of a test and to check the accuracy of an applicant’s analysis.
In addition, the paragraphs recanmend applicants to submit more analysis and
evaluation of data than before. EPA considers it appropriate for applicants,
in addition to Agency staff, to perform this work.
II. THE STPINDARDS FOR ACCEPTABLE TESTING
This part of the Discussion mcplains certain issues which have arisen
with respect to the standards governing the methodologies for performing
tests. The issues discussed here concern standards which would apply to the
performance of several kinds of tests. Issues involving technical aspects
of the standards which appear in only one kind of test are discussed in part
III of this Discussion.
A. Test Substance .
The “when required” paragraphs of the guidelines describe which prod-
ucts would have to be supported by data. The “test substance” paragraphs
prescribe what compound would have to be tested in order to generate the
required data. In many instances, data required to support the registration
of the pesticide product would be derived from tests performed with a sub-
stance which is a component of the product itself.
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The “test substance” paragraphs usually would require testing with
sane combination of the following three substances: the technical grade of
each active ingredient in the product, the manufacturing—use product, or
the end—use product. “Technical grade of the active ingredient” or
“technical chemical” refers to a particular chemical substance, the active
ingredient, having a certain level of purity, the technical grade. Presently,
the purity of the technical chemical is not fixed by an absolute standard.
It is usually determined instead by traditional industry methods, and
usually refers to that identifiable stable substance produced during the
normal manufacturing process which contains the highest concentration of
the active ingredient.
In all tests, the composition of the test substance would have to be
precisely identified. [ See 80—3(b)(2)(iv).] When the required test sub-
stance is a manufacturing-use product or end—use product, its composition
should meet or approximate the limits certified in accordance with
§ 62—2 of the Subdivision D product chemistry guidelines. In those cases
when the test substance does not conform with these limits, the Agency
shall make a determination as to the toxicological significance of the
deviations. The purposes and uses of certified limits are discussed in
n re depth in the Discussion on Subdivision D.
The lists below summarize the substances which would usually be tested
to satisfy the different data requirements:
Technical chemical :
Acute oral toxicity — § 81-1
Acute dermal toxicity - § 81—2
Acute inhalation toxicity - § 81—3
Acute delayed neurotoxicity — § 81-7
Subchronic oral toxicity — § 82-1 -
Subchronic dermal toxicity — § 82-2, —3
Subchronic inhalation toxicity — § 82—4
Subchronic neurotoxicity — § 82—5
Chronic toxicity studies — § 83—1
Oncogenicity — § 83—2
Teratogenicity — § 83—3
Reproduction — § 83-4
Mutagenicity — § 84—1 and 2
Special Studies — § 85—1 and 2 - An analytically pure grade of active
ingredient
Manufacturing—use product :
Acute oral toxicity — § 81—1
Acute dermal toxicity — § 81—2
Acute inhalation toxicity - § 81—3
Primary eye irritation - § 81—4
Primary dermal irritation — § 81—5
Dermal sensitization — § 81—6
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End—use product :
Acute oral toxicity — § 81—1
!/ Acute dermal toxicity - § 81—2
Acute inhalation toxicity — § 81—3
Primary eye irritation - § 81-4
primary dermal irritation — § 81—5
Dermal sensitization — § 81—6
in many cases a registered manufacturing—use product would be a technical
chemical. When this is the case, a single acute oral study using the prod-
uct would, for camp1e, satisfy the requirements to test both the technical
grade of the active ingredient and the manufacturing-use product. However,
when the manufacturing-use product is not a technical chemical, separate
studies with the product and the technical chemical would be required.
When test data would be used primarily as the basis for requiring
label warnings or special packaging, the Agency would require the product
itself to be tested. (In some cases, EPA would also require tests on use
dilutions.) Thus, all of the studies in Series 81 (acute toxicity and
irritation testing), except acute delayed neirotoxicity, 2/ would be per-
formed with the product for which registration is sought, either the manu-
facturing—use product or the end—use product.
The Agency uses data from acute studies on the technical grade of the
active ingredients to establish the relative toxicity of the chemicals, to
identify possible synergistic agents, and to evaluate the design of sub—
chronic tests. EPA considers the data on acute oral and acute dermal
LD5O, and acute inhalation LC50, to be basic toxicological data fran which
to begin the evaluation of any chemical.
The majority of the other tests in this subdivision are subchronic and
chronic studies, and the guidelines generally require that they be performed
with the technical grade of each active ingredient in the product. The
decision to routinely require intermediate and long term toxicity testing
only on technical chemicals is based both on the Agency’s experience in
reviewing the toxicity of the ingredients of pesticide products and on
econcrnic considerations.
!/ Use dilutions of the end-use product are tested in addition to the
formulation.
2/ The acute delayed nElrotoxicity study differs fran the other acute studies
in that the Agency would rarely impose the usual label warnings or special
packaging requirements as a result of data fran this study alone. Rather,
this study is used as a screen, and if positive results occur, further
testing is required. (See § 82—5.) Thus, acute testing for delayed ne ro—
toxicity is required routinely only for certain technical chemicals.
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Pesticide products contain many different kinds of ingredients. Every
pesticide contains at least one pesticidally “active .ingrediertt,” which by
definition produces toxic or behavior-altering effects in one or more kinds
of organisms exposed to the chemical. The ordinary commercial production
of pesticidally—active ingredients usually introduces manufacturing impurities
or byproducts which may also be toxic. Other chemical substances called
“inert ingredients” are defined by FIFRA 3/ as any ingredient in a product
which is pesticidally inactive. These substances, however, are not
necessarily non—toxic, nor are they necessarily biologically or chemically
inert. Some inert ingredients may be added to improve the usefulness of
the pesticide, to reduce its hazard to users, to dilute the chemicals, to
stabilize the ingredients, and for several other reasons. An “inert
ingredient” may bring its own impurities or may react with other ingredients
or packaging materials to produce new impurities. Finally, some pesticides
are somewhat unstable arid over time may partially degrade to form still
more substances.
EPA recognizes that any of the ingredients in a pesticide product
could be toxic to man or other beneficial organisms. Experience, however,
has shown that, with some notable exceptions, the active ingredients and
associated manufacturing impurities are of greatest concern. These
impurities, together with the active ingredient, make up what is usually
considered the “technical chemical.” Thus, testing the technical chemical
is consistent with that experience.
B. Number of Animals .
Each individual test section would specify a minimum number of animals
to be tee ted • The decisions on numbers of animals were be sed on the recom-
rnendations of experienced Agency and OECD scientists. Their- recommendations,
in turn, were based on a number of factors, including their knowledge of
the incidence of unusual and significant toxicological and pharmacological
effects, the level of statistical and technical confidence that each kind of
test should have for regulatory decision—making, and the relative cost of
larger and smaller numbers of animals. The Agency recognizes that increasing
the number of animals in any test may improve the sensitivity of the test.
But an increase would also raise the cost of the test. EPA considers the
number of animals to be a reasonable accommodation to the competing interests
in providing adequate sensitivity for the test systems and minimizing costs.
The statistical significance of test results is very important for
Agency decision—making. This is handled in two ways in the guidelines. In
the case of acute oral, dermal, and inhalation studies, the standards would
require that enough animals be used so that the LD5O or LC5O of the test
3/ See FIFRA Sec. 2(m)
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substance can be determined with a specified level of statistical certainty.
This approach is reasonable for these acute studies which are designed to
quantify a single toxic effect. It would be inappropriate for other tests
in which the Agency is concerned with a number of different toxic and
pharmacological effects. In eddition, the general principles governing all
testing provide that, if a toxicological or pharmacological effect occurs
with only marginal statistical significance, EPA may require that the test
be performed again with a larger number of animals. (See § 80—3(b)(lO)].
C. Range—Finding Studies and Selection of Dose Levels .
The principle concerning selection of dose levels generally specify
that the chosen levels would have to produce a particular result. For
example, § 83—3 would recommend that the highest of the three dose levels
in a teratogenicity study must produce some toxic effect in the treated
mothers.
The recommendation that the highest dose level would have to produce some
toxic or pharmacological effect appears in several sections, and is designed
to ensure that the test is performed at a sensitive level. (However, EPA
may accept data from a study which appears valid even when an extremely
high dosage level fails to elicit a toxic response.)
Each subchronic section recommends that the highest dose level be chosen
to result in definite but not excessive toxicity. The Agency feels that
the high dose level should be chosen to characterize the maximally tolerated
dose rate for these exposure periods in order to allow sufficient spread
between the lowest and intermediate dosage ranges to obtain good dose—
response information.
A number of sections also contain a principle that the lowest dose
level should produce no evidence of toxicity. The requirement for a “no
observed effect level” comes primarily from EPA’s tolerance—setting
procedures under the Federal Food, Drug, and Cosmetic Act (21 USC 321 et
seq.) 4/. To set a tolerance with an ample margin of safety, the Agency
calculates an acceptable daily intake by dividing the “no observed effect
level” for a pesticide by an appropriate safety factor which depends upon
the type of study and the toxic effect observed. Residues of that pesticide
in food or feed up to the tolerance level are permissible. Similar consider-
ations affect the Agency’s decisions to approve certain other kinds of use
patterns for pesticides.
4/ This Discussion describes the Agency’s tolerance testing proc ss only
briefly. A n re comprehensive explanation of the mechanics of estab-
lishing tolerances can be found in EPA’s “Tolerance Paper” which may
be requested from EPA. See also part X lv of the Discussion on Sub-
division M for the FDA 1968 guidelines on residue tolerances.
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As noted above, EPA ordinarily uses the no observed effect levels
established in various studies in making regulatory decisions concerning
safe levels of human exposure. Some use patterns, however, may involve
accidental inadvertent human exposure at levels higher than the no observed
effect level. To evaluate risk in these situations, EPA would need data
from dose levels substantially higher than the anticipated level of human
exposure. If the dose levels of an available study would not give the
Agency adequate information to approve a particular use, EPA would require
that additional testing be conducted.
The Agency recognizes that in order to satisfy these principles, appli-
cants may find it useful to perform a range—finding test before beginning
the full scale study. The Agency also recognizes that these preliminary
tests, which generally use fewer animals and nvre dose levels than required
by the current guidelines, can yield other valuable information helpful
to the design of the full scale study. These guidelines, however, would
not require applicants to perform this preliminary testing, both because
sueh tests are not always needed and because the appropriate design for a
preliminary test depends on what information is already known. Consequently,
it would be difficult to specify in the guidelines when and how range-finding
studies should be conducted.
D. Control Groups .
Most of the sections contain studies which would require control
groups. Control groups are normally included in biological tests to deter-
mine whethei any observed effects are attributable to exposure to the test
substance. For the purposes of this subdivision of the guidelines, there are
three important types of control groups: untreated (negative) controls, vehicle
controls, and positive controls. The animals of any of these three control
groups would have to be the same age and come from the same source as the
comparable animals receiving the test substance. In addition, to prevent
any possible bias, animals chosen for the test and control groups would
have to be assigned at random. Finally, the control groups recommended
by the guidelines would, of course, have to be run concurrently with the
groups receiving the test substance.
A vehicle control group would be recommended for nearly all studies.
The studies would require animals in the vehicle control group to be
treated in a manner identical to the test groups in every respect except
for exposure to the test substance. They should receive the same care,
diet, and ancillary materials (e.g., vehicle or dust suppressant) and be
subjected to the same environmental, conditions as the animals exposed to
the test substance.
some cases, the studies would have an untreated (negative) control
group, in addition to the vehicle control group. A negative control group
is usually necessary to determine whether any effects observed in the
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vehicle control or test group animals are due to the n difying effects
(e.g., synergism, antagonism, or independent toxicity) of the ancillary
materials (vehicles) used in the study. Or, if no vehicles are used, the
negative control operates as a direct comparison with the test groups used
in the study. Thus, animals in the negative control group would be treated
like the vehicle control group and test group animals, except that they
would receive neither the test substance nor any of the ancillary materials
(e.g., vehicle or dust suppressant).
positive control group, when used, serves as an internal quality
control: to ascertain whether the test substance produces an effect similar
to a related substance of known toxicity; to ascertain whether laboratory
staffs are properly making and recording sophisticated visual determinations
and properly carrying out other aspects of the test; and to ascertain if
a strain or species reacts similarly to another species or strain when ex-
posed to a known standard toxicant. For example, in delayed neurotoxicity
testing, triorthocresylphosphate (TOCP) may be used as a principle against
which to measure the neurotoxic effect of a substance. The Agency believes
that the use of a positive control with each set of test groups would be
quite important for three kinds of studies (acute delayed neurotoxicity,
subchronic neurotoxicity, and mutagenicity) due to the sophisticated
observations and diagnoses involved.
Data on a fourth kind of control group, historical or colony controls,
are also sometimes used in evaluating test results. Historical control data
usually give the investigator information about the longevity, fecundity,
or incidences of spontaneous tuners and other diseases of one species or
strain of animal. Comparison to vehicle or negative control data may also
indicate whether the test animals are typical of their species and strain.
From a scientific viewpoint, however, it is not satisfactory to rely solely
on historical control data for accurate control information. Since histor-
ical controls are not run concurrently with the testing of the substance
under study, the environmental conditions of the study may be different; the
animals may have come from a different breeding colony; or the measurements
may have been obtained at different times of the year. Any of these (and
many other) differences could affect the reliability of any comparisons be-
tween historical controls and test groups. Yet historical data is valuable
for certain comparative or statistical purposes, and such data should be
submitted when pertinent, or upon Agency request.
The Agency realizes the scientific benefits of vehicle and negative
controls run concurrently with the test groups. It also recognizes that
both of these control groups may not be necessary for each of the tests
proposed in the guidelines. Positive control data, too, are useful, pri-
marily for internal quality control. EPA recognizes, however, that some
of the substances used as positive controls may expose the testing personnel
to undue risks and create disposal or environmental safety problems.
Using concurrent control groups also increases the number of animals
and analytical procedures recommended, and therefore increases the cost of
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each test performed. In the current guidelines, the Agency has accordingly
attempted to recanmend the various kinds of control groups as prudently as
possible. (See § 80—3(b)(5).)
E. Clinical Laboratory Tests .
Clinical laboratory tests of animals are proposed in a number of
studies. (See, e.g., § 82-1, 83—1.) Most frequently, EPA would recan—
mend some measurements on blood and urine samples, though occasionally
other data, including food consumption and body weight, are requested.
EPA considers that data developed from clinical tests give a better
indication of the onset and development of toxic effects than can be gained
from observation of the animals’ behavior alone. In addition, effects
detected in clinical tests often can support data generated through post-
mortem examination of test animals. Accordingly, EPA believes that some
clinical testing is necessary.
F. Observation and Handling of Animals .
All of the guidelines would recommend procedures for observation of
test animals. The provisions often establish two criteria relating to
frequency of observation. First, appropriately—trained personnel would be
expected to observe the animals as frequently as needed to detect behavioral
or other observable expressions of toxic effects on an animal. In the
case of short-term studies, EPA expects that such observatio-ns would occur
at least twice a day. Daily observation would be adequate in most cases
for studies lasting several weeks or longer. In all studies, however,
investigators would be expected to observe animals more frequently if
necessary to detect signs of toxicity.
Second, personnel would be expected to observe animals as frequently as
necessary to prevent loss of the animals by cannibalism, autolysis, misplace-
ment, and similar management problems. The guidelines specify that sick
animals should be moved to separate cages, and moribund animals should be
sacrificed. These procedures should help to assure that statistically
sufficient numbers of test and control animals are used for the purpose of
generating the necessary data.
The principles paragraphs also state that all signs of toxicity observed
throughout a study would have to be recorded and reported. In response to
early comments that this provision alone was too vague to be useful, EPA
has added a list of toxic signs sthith should be recorded, if observed.
This list, however, is not exhaustive, and any other signs noted would also
be required to be reported.
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G. Extent of Necropsy and Histopathology .
Many of the guidelines sections recaiunend that at least some of the
animals in the study be subjected to gross necropsy and his topathology
examination. A necropsy involves dissection of an animal and examination of
its tissues for grossly visible indications of toxic effects, such as, for
example, an enlarged liver. Necropsy often includes removal and weighing
of certain organs, as well. A histopathology examination involves micro-
scopic examination of slides containing tissue frce different parts of an
animal’s body. A histopathology examination will detect changes at the
cellular level, such as cancer or degeneration of nerve fiber. To perform
necropsies or histopathology examinations properly requires considerable
training, skill, and time. Consequently, these procedures are costly, and
there are only a relatively limited number of people who are qualified to
perform them. These procedures, however, are also a very reliable way of
identifying and measuring the toxic effects caused by a pesticide. EPA is
concerned with the development of principles which will generate all of the
useful information which can reasonably be obtained from necropsies and
histopathology examinations without overburdening the nation’s testing
resources with expensive recosunendations.
The principles governing histopathology examinations in subthronic and
chronic studies are also generally designed to minimize the number of
animals which must he examined while maintaining the sensitivity of the
studies. These principles are based on the assumption that, especially in
short—term studies, most toxic effects follow a clear dose—response pattern;
the incidence and severity of the effect increase at higher dose levels.
Accordingly, EPA would recosimend in subthronic rodent studies that the tissues
of all high dose level and control animals be examined microscopically. 5/
The liver, kidney, and lungs would have to be examined in the intermediate
and low dosage level groups, but if no canpound—re].ated effects are observed
in any other tissue in any high level animal, then such other tissues would
not need to be examined microscopically in animals from the lower dose
levels. If, however, effects are observed at the high dose level, the
tissues showing the effects would have to be examined in all animals in the
lower dose levels.
5/ Section 83-1, chronic toxicity study, includes principles for a test
using four non—rodents per sex at each dose level. All non—rodents
from each dose level- ust be subjected to a histopathology examination.
Non—rodents would be treated differently from rodents in this study
because far fewer animals are involved, and because this is the only
major study to use non—rodent mammals.
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Somewhat more extensive histopathology examinations would be recan-
mended by the chronic toxicity studies, § 83—1. Occasionally, histo—
pathologic effects may not be manifested at the highest dose level,
particularly when that level causes significant growth depression; these
effects would therefore likely appear at the next lower level. In addition,
unlike the younger animals used in subthronic s tudies, old animals examined
at the end of lifetime studies often show a great deal of spontaneous
disease involving multiple organ systems. Chemically—induced lesions,
intermixed with the effects of disease, may be missed during the gross
necropsy. Because of these possibilities, EPA thinks a chronic feeding
study should include a more extensive histopathology examination than a
subthronic study.
There are two other qualifications to the general approaches described
above. First, any tissue from any dose level showing evidence of an effect,
either fran necropsy or clinical test results, would have to be examined at
all dose levels. Second, those tissues which are most often affected by
toxic pesticides would have to be examined from every dose level. EPA
onsiders that following these standards will generally detect most toxic
effects and minimize the number of animals which would be examined.
Necropsy and his topathology in the Oncogenicity Study and Teratogenicity
Study, § 83-2 and —3, are handled differently fran the other chronic and
subchronic studies. No histopathology examination would be expected for
teratogenicity testing because most birth defects (terata) are grossly
visible in animals at necropsy. Necropsy would be recommended on all
animals, since the high dose, which should cause toxic effects in the
mothers, may mask the appearance of birth defects. In an oncogenicity
study, all animals fran all dose levels would be subject to necropsy and
his topathology examination, because data from a number of tests have shown
that a stronger oncogenic response may be elicited fran a lower dose level
than from the highest dose level. This anomalous dose—respo nse pattern may
be attributable to dose-related differences in phartnacokinetics and metabolism
of the chemical or to cellular or tissue destruction at he higher dose
level (resulting, in many cases, in early mortality) which may prevent the
same level of response that would be elicited at the lower levels.
III. DISCUSSION OF INDIVIDUAL TESTS
This part of the Discussion explains the purposes for requiring data
from each of the different kinds of tests and the major issues identified
in developing individual guidelines sections.
A. Acu1 Oral, Dermal, and Inhalation Toxicity; § 81-1, -2, —3 .
Data from acute oral, derinal, and inhalation toxicity studies are used
for a number of purposes • EPA considers these data in de termining the rela-
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tive toxicity of various compounds, how a pesticide causes toxic effects,
and the proper design of longer—term studies. In addition, a number of speci-
fic regulatory decisions are based on acute toxicity data. The decisions
deal with determinations as to whether to: issue a rebuttable presumption
ageinst registration (RPAR) (40 CFR § 162.11(a)]; classify a pesticide for
general use or restrict it for use only by certified applicators (40 CFR
§ 162.11(c)]; require special packaging (as proposed in 40 CFR § 162.16); and
require that the pesticide product labeling contain certain hazard warnings
(40 CFR § 162.10). The kind of hazard warning depends on the toxicity cate-
gory to which a pesticide belongs. The categories are ranges of toxicity
determined at either end by an LD5O or LC5O.
The oral, dermal, and inhalation toxicity guidelines sections contain
principles which provide that if no mortality is produced by administration
of a specified dose level, no further testing is required. (See “Limit
test” paragraphs.) In the acute oral and inhalation toxicity guidelines,
these dose levels correspond to the lower limit of Toxicity Category IV.
No matter how much higher the LD5O or LC5O, the substance will be placed in
Category IV, and no other regulatory action will be taken on the basis of
the product’s acute toxicity. In the acute dermal toxicity guidelines
section, however, the upper dose level is 2 g/kg, rather than the lower
limit for Toxicity Category IV, 20 g/kg. Agency staffs believe that it
would be physically impossible to administer a larger dose to rabbits.
(It is noted that the Agency is currently in the process of revising its
pesticide product labeling regulations to reflect the physical impossibility
of dosing at greater than 2 g/kg.) Moreover, Agency staffs are persuaded
that no human would be likely to absorb as much as 20 g/kg (approximately
2 1/2 lbs. for the average adult American) of any substance by the dermal
route, and that 2 g/kg is a more reasonable approximation of the largest
dose which a human could possibly absorb through the skin.
B. Irritation and Sensitization Studies; § 81—4, —5, —6 .
Data from three studies, primary eye irritation, primary dertnal irrita-
tion, and dermal sensitization, are used principally to support label pre-
cautionary statements. Eye and dermal irritation data, just as acute oral,
dermal, and inhalation toxicity data, are used to place a product within a
toxicity category. Section 162.10 of the registration regulations requires
a product’s label to bear particular hazard warnings depending on the
products toxicity category. Positive results in a dermal sensitization
study, too, will result in requirements for label warnings.
C. Neurotoxicity Evaluations; § 81—7 and 82-5 .
A neurotoxic pesticide is one which causes damage to the nervous system.
Neurotoxicity can occur in many different ways, and, therefore, most guidelines
sections require an applicant to look for signs indicating that a pesticide
may affect the nervous system.
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These guidelines also include two sections which focus exclusively on
neurotoxicity. The first, § 81—7, is a special type of test designed to
screen for only one of the many different kinds of neurotoxicity, “delayed
neurotoxicity.” Delayed neurotoxicity is a syndrome typically caused by
certain organophosphate chemicals in which damage to nervous system causes
unsteady reflexes and eventually can result in paralysis. These signs
first appear several days to a few weeks following exposure, and hence the
effect is “delayed.” EPA’s experience indicates that every compound which
produces delayed neurotoxicity in any test system will also cause delayed
neurotoxicity when given in a very large dose to hens under an acute regimen.
Thus, the acute delayed neurotoxicity study would be used to identify
these compounds, and if positive results are observed in the study, further
testing would be required.
The subthronic guideline section 82-5, was designed primarily for further
testing of substances which have been shown to produce organophosphorus—like
delayed neuropathy. The test animal is the hen.
Alternate procedures may be required if a substance other than an
organophosphorus material is to be tested for delayed neuropathy, or if a
substance is to be tested for a neuropathy other than the organophosphorus-
type delayed neuropathy. In such situations usually the test animal will
not be the hen. Instead, the species in which the neuropathy had been
demonstrated might be used. Testing of both sexes might be required.
Such an altered test in which a mammalian species is used might be
combined with another subthrortic study, pxovided the purposes of both
studies were satisfied.
When a no—observed-effect level for a neiropathy is to be determined
and an altered or different procedure is to be used, prior consultation
with EPA scientists is advised.
D. Subchronic Studies; § 82—1, —2, —3, and —4 .
Subchronic studies involve regular repeated exposure to a pesticide,
and data from at least one of these studies would be required to support
the registration of virtually every product. Data from these studies serve
as a bridge between information developed in acute and chronic tests. Two
important purposes of subchronic testing are to identify target organs and
to establish appropriate dose levels for lifetime studies. In addition,
the subchronic studies are often used to evaluate the relative toxicity of
a substance using different routes of exposure. Finally, the clinical
testing which would be required in subchronic studies is considerably more
extensive than that which would be required in chronic studies. These
additional data are useful in understanding the development of toxic
effects.
This subdivision also contains two different sections describing sub-
chronic dermal toxicity studies. The Repeated Dose Dermal Toxicity: 21—Day
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Study, § 82—2, is considerably less extensive than the Subchronic Dermal
Toxicity: 90—Day Study, § 82—3. Data from the 21—day study is essential to
support the registration of all products whose use is likely to result in
repeated human skin contact. This study would be used primarily to assess
subchronic local dermal toxicity, that is, effects on the skin at the site
of application. Some data on systemic toxicity are also produced by this
study; these data would be compared with any available data from a subchronic
oral toxicity study to determine whether the pesticide is potentially more
toxic by the derinal route.
Data from the 90-day dermal study is essential when human dermal expo-
sure is purposeful, and either the dermal route of exposure appears to be
more toxic than the oral route or no subthronic oral data are necessary.
If these conditions exist, the Agency considers it necessary to recommend an
extensive subthronic dermal study of systemic toxicity. Thus, this section
would extend the duration of dosing to 90 days, increase the clinical best-
ing recommendations, and expand the scope of the necropsy and hi.stopathology
examinations beyond the standards proposed in the 21-day study. The scope
of the 90-day derinal study, then, would be comparable to that of the rodent
subchronic oral toxicity study.
E. chronic Toxicity Studies and Oncogenicity Studies; § . 83—1, —2, 5 .
This subdivision includes three sections which describe tests for
detecting the adverse effects resulting from long—term exposure to a pesti-
cide. One of the sections, Oncogenicity Studies ( 83—2), focuses on the
detection of malignant and benign tumors and preneoplas tic lesions. Another,
Chronic Toxicity Studies ( 83—1), is designed primarily to evaluate other
thronic effects. (While the route of administration of the test substance
is generally oral, the study is required to support the registration of
products whose use resulted in repeated human exposure by any route.)
Nonetheless, despite minor differences in design and purpose, both studies
are capable of detecting oncogenic and non—oncogenic effects, and both
kinds of effects must be reported, regardless of the study in whid they
appear. Moreover, because the studies are so similar, they usually can be
combined into a single test when the rat is the test species for both.
Because of this, s tandards for a combined s tudy to meet the requirements of
both sections has been added at § 83—5, Combined Chronic Toxicity/Oncogeni-
ci by Studies • Such a canbi ned s tudy provides for the evalua tio n of exposure
to the test substance in rodents covering the majority of the expected life
span of the strain (approximately 24 it nths duration in rats and 18 itonths
in mice). This is consistent with EPA’s policy concerning “food use
pesticides,” and is based on the Agency’s experience in reviewing data
from chronic feeding studies in rodents and therefore will remain in effect
until further notice.
EPA thinks that use of a non-rodent species in thronic toxicity studies,
as well as a rodent species, is necessary to provide an adequate evaluation
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of non—oncogenic chronic effects (see § 83—1). The non—rodent, usually
the dog, may metabolize the test substance differently fran the rodent,
and therefore the two species, together, may reveal a much broader range
of toxic effects than if only one species were tested. (See, generally,
Goldenthal, E., “Current Views on Safety Evaluation of Drugs,” FDA Papers,
13 (May, 1968); Fitzhugh, 0.0., 1965, “Appraisal on the safety of chemicals
in foods, drugs, and cosmetics — chronic oral toxicity,” pp. 36, 38.
Assoc. Food & Drug Of fic. of the U.S., Topeka, Kansas.]
EPA believes that conducting oncogenic and chronic feeding studies for
predetermined long—term periods is valuable for several reasons • EPA thinks
that these time periods are long enough to allow tumors and other chronic
effects to develop, and yet short enough to assure that a reasonable
percentage of the animals will survive to the scheduled termination point.
A high level of survival would provide the cperimenter with a larger data
base on which to perform statistical analysis. In addition, since geriatric
diseases often make diagnosis of neoplastic growth difficult, the studies
would be designed to produce meaningful histology by terminating the studies
before such diseases normally becane a significant problem. By using a
relatively uniform duration, EPA would also be better able to canpare the
results of different studies and to canpare results with the historical
data base. Finally, the principles establish a fairly definite endpoint for
chronic studies which would allow experimenters to plan their studies more
efficiently.
F. Teratogenicity and Reproduction Studies; § 83—3, —4 .
Two studies in the chronic/long—term series are designed to evaluate
the effects of a pesticide on the process of reproduction. Data fran a
Teratogenicity Study, § 83—3, indicate whether exposure to a pesticide dur-
ing pregnancy can cause fetotoxicity or birth defects in offspring. The
Reproduction/Fertility Study, § 83-4, involves exposure to a pesticide
arid is designed to assess more subtle effects on reproduction, such as
decreased fertility, premature delivery, and smaller offspring.
Commenters have suggested that, in addition to the Teratogenicity Study
in § 83—3, tests on behavioral and central nervous system (CNS) defects may
be useful. Evaluation of defects in the CNS would have to be done in
litters during the postnatal period. The need for postnatal teratological
evaluation arises mainly because the CNS does not mature fully in the human
fetus and may still be liable to certain teratogenic influences in late
pregnancy. The studies would involve treating pregnant female animals during
the latter third of gestation, observing the neonate throughout sexual
maturity, and perhaps performing objective tests on these animals, if
appropriate. Comments are encouraged on the characteristics of a suitable
test species, number of test animals, dose levels, duration of neonate
observation, and tests to be performed on these neonates.
The principles for the reproduction study represent a significant depar-
ture from traditional testing procedures. Instead of a three-generation
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study with two litters in each generation, EPA provides a two-generation
study with only one litter in each generation. The Agency considers that
this methodology is generally more sensitive than the traditional test
design. In addition, this methodology is considerably less expensive than
the traditional test design because fewer animals are required.
A traditional reproduction study requires two litters per generation
because the first litters produced by adolescent mothers often show a great
deal of variation. Because of these variations, data based on the first
litters are less useful than data from the second set of litters, which are
more uniform in size and health. The current guidelines recommend only one
litter, but the animals would not be bred until they had fully matured.
Consequently, under the current principles, the first litter would be less
likely to show the variability of the first litters produced by juvenile
mothers in a traditional study.
In addition to reducing the number of litters per generation, this
guideline would reduce the number of generations. Part of the reason
reproduction studies traditionally have tested three generations is to
detect genetic abnormalities. Under the Subdivision F guidelines, however,
the potential of a pesticide to cause genetic damage would be assessed in
a battery of mutagenicity tests which are collectively more sensitive than
than a three—generation reproduction study. (See also part III. G. of this
Discussion.) The other reason for extending a reproduction study into the
third generation is to detect cumulative effects. The Agency, however, is
not aware of any toxic effect which first appeared in the third generation
of a reproduction study. While the current guidelines reduce the number of
generations, it would extend the period of dosing for each litter-producing
animal. Accordingly, EPA believes this test design to be more sensitive
for cumulative effects than the traditional three—generation study.
G. Mutagenicity Studies § 84—1 and —2 .
Data from mütagencity tests are used for several purposes. Data are
used directly to determine if a test substance is genotoxic, that is,
capable of interacting with or damaging genetic material (DNA) and/or
mechanisms, or mutagenic, and hence capable of producing heritable genetic
changes. The Agency recognizes that a mutagen causing a heritable defect
in humans has not yet been identified, however the design and conduct of
an epidemiologic study to verify such a relatively rare event is difficult.
Since mutations do occur in humans, the Agency feels that it needs to
collect information in experimental test systems in order to make reason-
able decisions regarding risks associated with chemicals which may be
capable of inducing human genetic alteration. Mutagencity data may also
be used as an indication of the oncogenic potential of a pesticide.
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After considering the public comments to the mutagenicity testing
requirements in the 1978 FIFRA proposed guidelines (40 CFR 163)
the Agency has decided to allow more options in satisfying test
eqUiramefltS The different tests vary in their ability to detect
mutagens depending on the chemical structure or the physical
properties of the test substance. In addition many of these tests
are still being validated and consequently individual laboratories
may prefer to perform assays with which they feel more confident.
The Gene—Tox Program of the EPA Office of Toxic Substance (OTS) has
made recommendations on major mutagenicity tests and protocols are
being finalized for the test standards of o’rs and for the international
testing programs of OFCD. Other protocols may be found in the
EPA/SRI International Project “In Vitro Mutagenicity Studies of
Environmental Chemicals,” as well as in the Report of the International
Collaborative Program “Evaluations of Short-Term Tests for Carcinogens”
(Progress in Mutation Research vol. 1, ed. by F.J. de Serres and
J. Ashby, Elsevier/North Holland, New York, 1981)
In view of these ongoing efforts, the mutagenicity testing subdivision
of this document describes the criteria for selecting a battery of
tests and lists representative tests which may be acceptable for this
battery. Additional tests not listed may also be acceptable if
sufficient documentation is furnished for the Agency to validate
their usefulness.
For each test substances a battery of tests is required to assess
potential to affect the qualitative or quantitative integrity of
human genetic material. The objectives underlying the selection
of a battery of tests for mutagencity assessment are:
1. To detect, with sensitive assay methods, the capacity of a
test substance to alter genetic material in cells,
2. To determine the relevance of these changes to mammals; and,
when mutagenic potential is demonstrated,
3. To incorporate these findings in the risk assessment for
heritable effects, onconc nicity, and possibiy, other health
endpoints.
The battery must include tests appropriate to address the
following three categories of genetic effects:
(1) gene mutations
(2) structural chromosomal aberrations
(3) other mechanisms of mutagenicity (e.g. spindle inhibition,
direct DNA damage) as appropriate for the tested chemical.
Specific battery test selection and protocol desigo should be
suhnitted to the Agency for comment and evaluation. Registrants
are encouraged to discuss results of preliminary testing with the
Agency. -
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§ 85—2 Domestic Animal Safety Testing .
Data from Domestic Animal Safety Studies are to be derived from studies
using specific domestic animals (e.g., cat, dog, chicken, cow, pig) as test
animals for the various kinds of tests required in this subdivision of the
guidelines, such as acute oral, subchronic inhalation, etc. Such data would
be required on a case-by-case basis when the Agency cannot satisfactorily
evaluate how a pesticide will affect certain domestic species by using data
otherwise required by the subdivision. EPA may require such data, for
example, when a pesticide, such as a tick collar or louse dust, is being
used directly on a domestic animal. When the Agency decides additional
data are needed, it will also establish appropriate principles for accep-
table testing to generate the data.
§ 85—3 Derrnal Absorption Studies of Pesticides .
The skin can be the major route of human exp ure to pesticides
during application, harvesting and home use; however, dermal absorption
studies are not considered routinely necessary. Derrnal absorption
studies may be required on an individual basis for compounds having a
serious toxic effect, identified by oral or inhalation studies, for
which a significant route of human cposure is derinal and for which the
assumption of 100% aborption does not produce an adequate margin of safety.
Registrants should work cl ely with the Agency in developing and
performing dermal absorption studies. The experience of Agency scientists
has identified many problems In published dermal absorption studies which
make them inappropriate for pesticides. Derrnal absorption protocols
developed by the FDA should not be used for pesticides since they are
designed specifically for the types of formulations controlled by the FDA.
The Agency has available, on request, a protocol for in vitro
determination of dermal absorption. This protocol is undergoing practical
evaluation in the laboratories of several registrants but It is not
considered ready for publication. The Agency is also interested in in vitro
methods but the Agency scientists involved do not consider themselves ready
to write a protocol. Practical suggestions based on hands—on experience
in in vitro methodology are being sought fran individual investigators.
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SUBDIVISION F -- HAZARD EVALUATION: HUMANS AND DOMESTIC ANIMALS
Series 80: OVERVIEW, DEFINITIONS, AND GENERAL REQUIREMENTS
(NOTE: The sections in this series are essentially the same as those pub-
lished in the 1978 proposed guidelines for Subpart F. The current sections
offer important and useful guidance to those engaged in developing toxicology
data to meet pesticide registration requirements.
IMPORTANT: If the recommendations of any section in this Series 80
differ from those of the specific test sections of Subdivision F or the
good laboratory practice requirements of 40 CFR Part 160 (soon to be
published) then the latter shall be followed.]
80—1 Overview .
This subdivision details the toxicity data recommended to support the
registration of pesticide products. Each section specifies the conditions
under which the data are required. These data are evaluated to determine
potential adverse toxicological effects to humans and domestic animals as
a result of use of a pesticide. Having made these evaluations and determin-
ations, the Agency must then determine whether:
(a) The application for registration should be approved (see § 162.7(d)
of the FIFRA sec. 3 regulations]; -
(b) The pesticide gives rise to a rebuttable presumption against
registration (see § 162.11(a)];
(c) The pesticide is a candidate for general or restricted use classi-
fication (see § 162.11(c)];
(d) The labeling of the product is adequate to protect field workers
and applicators and complies with the requirements of FIFRA (see § 162.10);
and
(e) The pesticide product is subject to special packaging requirements
(see § 162.16).
§ 80—2 Definitions .
(a) Terms used in this subdivision shall have the meanings set
forth at § 162.3 of FIFRA sec. 3, at § 60—2 of Subdivision D, and at
§ 190-2 of 40 CFR Part 160.
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(b) In addition, for the purposes of this subdivision:
(1) The term “pharmacological effect” means any chemically—induced
physiological thange in a test animal.
(2) The term “target organ” means any organ of a test animal showing
evidence of an effect of chemical treatment.
Cc) Refer to the individual test sections of this subdivision for
definitions of additional terms.
§ 80-3 General provisions .
(a) Scope . The standards contained in this section apply to all
studies in this subdivision unless another section of this subdivision
contains a specific standard on the same subject. In such a case, the
specific standard in the other sections should apply to the conduct of
that particular study.
(b) Basic principles for testing .
(1) Personnel .
Ci) All testing and evaluation must be done under the direction of
personnel who have the education, training, and experience to perform
the testing and evaluation in accordance with sound scientific cperimental
procedures. The Agency may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing.
(ii) To help assure consistency in the development of data, one
person should be responsible for each particular phase of a study. This
is especially important with respect to the conduct of necropsies, when
several persons are assigned separate tasks in a necropsy procedure.
(A) A Board-Certified or Board—Eligible pathologist or a person
with equivalent training, with experience in laboratory animal pathology,
should have the final and overall responsibility for all necropsy and
histopathology procedures, and for the accuracy and reliability of all
diagnoses, conclusions, and reporting.
(B) A properly trained pathology assistant, under the direct super-
vision of the pathologist, may perform gross necropsy.
(C) A histology technician, such as one certified by the P 1 merican
Society of Clinical Pathology (HTASCP) or one having equivalent training
and capability, may be responsible for all histologic preparations.
(iii) An appropriately educated, trained, and experienced toxicologist
should be ultimately responsible for the execution of all phases of each
study.
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(2) Test substance .
(i) Sections 81—1 through 85—2 specify ether the data submitted in
support of an application for registration shall be derived from tests
conducted with the technical grade of the active ingredient, the end—use
product, both, or some other substance.
(ii) The technical grade of the active ingredient is often the same
substance as the manufacturing—use product. In this case, where the sections
require testing of the technical grade of the active ingredient, a sample
of the manufacturing—use product shall be tested. Where this is not the
case, the tests shall be conducted with the technical grade of the active
ingredient thith is used to produce the manufacturing—use or end—use
pesticide product.
(iii) The lot of the substance tested should be the same throughout
the duration of the study, and the research sample shall be stored
under conditions that maintain its purity and stability. If the stability
of the test substance cannot be maintained for the duration of the study
or if, for other reasons, it is not possible to use the same lot throughout
the test, subsequent lots of the test substance can be selected that are
as nearly identical to the original lot as practical. Chemical assays
shall be performed to assure this identity and permit reporting of any
deviation in composition.
(iv) The composition of each lot of the test substance shall be
determined, including the name and quantities of known contaminants and
impurities, as far as is technically feasible. The determination shall
include quantities of unknown materials, if any, so that 100 percent of
the test sample is accounted for. The test substance shall be within the
limits, if any, certified in accordance with § 62—2.
(v) If the test or control substance is to be incorporated into feed
or another vehicle, the period during thith the test substance is stable
in such a mixture shall be determined prior to the start of the study.
No mixture of test or control substance with the feed or vehicle shall be
maintained or used during a period exceeding the known stability of the
test or control substance in the mixture. Alternatively, determinations
of the stability of the test or control substance in statistically randomized
samples of the diet or vehicle mixture shall be made periodically during
the study to ensure that proper mixing, formulation, and storage procedures
are being followed and that the appropriate concentration of the test or
control substance is contained in the mixture. (See 40 CFR Part 160, Good
Laboratory Practices.)
(vi) If the best substance is incorporated into feed or another
vehicle, its homogeneity and concentration shall be determined prior to
the start of the study and periodically during the study (40 CFR Part 160,
Good Laboratory Practices). Statistically randomized samples of the
mixture sh al 1 be analyzed to ensure that the proper mixing, formula tb n,
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and storage procedures are being followed, and that the appropriate concen-
tration of the test or control substance is contained in the mixture.
(vii) In addition to or in lieu of data otherwise requested by this
subdivision, the Agency may reguest, after cor ultation with the applicant,
data derived from testing to be conducted with:
(A) An analytically pure grade of an active ingredient;
(B) The technical grade of an active ingredient;
(C) An inert ingredient of a pesticide formulation;
(D) A contaminant or impurity of an active or inert ingredient;
(E) A metabolite (fr i animals or plants) or degradation product of
an active or inert ingredient;
(F) The pesticide end—use product.
(G) Any additional substance which enhances the toxic activity (up
to and including synergistic effects) of the product for which registration
is sought; or
(H) Any canbination of the best substances mentioned in paragraphs
(b)(2)(vii)(A) through (G) of this section.
(3) Administration of substance and vehicles .
(i) The manner of administration of the test and control substance
should be selected so as to maintain accuracy of the dosage.
(ii) All doses in a study should be administered to the animals by
the same route and method.
(iii) Where dosing is daily, dosing treatments should be conducted
at approximately the same time each day.
(iv) If a vehicle is used to dissolve or dilute the test substance
or positive control substance, it should be chosen to possess the following
characteristics to the greatest degree known:
(A) It does not alter the absorption, distribution, metabolism, or
retention of the test substance;
(B) It does not alter the chemical properties of the test substance
or enhance, reduce, or alter the toxic characteristics of the chemical;
(C) It does not affect the food and water consumption or the nutri-
tional status of the animals;
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(D) At the levels used in the study, it does not produce physiological
ef fects; and
(E) It clc8ely resembles the vehicle, if any, to be used under pected
conditions of use.
(4) Control groups . Control groups are used to assure that effects
observed are associated or attributed to the test themical exposure. The
appropriate control group shall be identical in every respect to the test
group accept for exposure to the test substance. Within a given study,
all control animals shall be frost the same source, be of the same age,
receive the same care, and be fed from the same batth and lot during the
same period as the animals receiving the test substance. To prevent bias,
a system to randomly assign animals to test groups and control groups is
required. [ See also paragraph (b)(5) of this section.]
(i) Untreated (negative) control group . An untreated control group
is usually required. This group receives neither the test substance
nor any ancillary material (vehicle). Consult individual sections of this
subdivision for those tests where an untreated control is require.
(ii) Vehicle control groups . (A) If a vehicle is used to administer
the test substance, a concurrent vehicle control group is recanmended.
Animals in this group receive treatment with the vehicle alone, usually at
the highest level the vehicle is used for any test group in the study.
Consult individual sections of this subdivision for those tests where a
vehicle control is required.
(B) As provided in paragraph (b)(3)(iv) of this section, the vehicle
shall be selected on the basis of information establishing that it is
non—toxic at the levels used in the study, has no independent physiological
effects, and does not alter the themistry or toxicity of the test substance.
If, however, there are insufficient data on the effects of the vehicle,
testing of the vehicle is required.
(iii) Positive control group . Positive control groups generally are
not recciiunended. These groups serve as an internal quality control, to
demons tr a te wh e the r the tes t animals are se ns i tive to or respond in a
predictable manner to known toxic agents, and to ascertain if a strain or
species reacts similarly to another strain or species when cposed to the
same known or standard toxicant. Consult individual sections of this
subdivision for those tests where a positive control is recccnmended.
(iv) Historical (colony) controls . Data on historical controls are
required when the Agency desires information on the longevity, spontaneous
diseases, and tumor incidences of a species or strain selected for a study,
and for certain cc*nparative or statistical purposes. Consult individual
sections of this subdivision for those tests where historical control data
are required.
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(5) Animal care and selection .
Ci) Each animal shall be assigned a unique identification number.
(ii) All data su titted in support of an application for registration
must be derived from tests conducted in accordance with good laboratory
practices. Healthy animals shall be used, and kept in conditions conforming
to od husbandry practices. Animals shall be assigned to test groups in
such a manner as to minimize bias and assure comparability of pertinent
variables. The animals of all test groups shall, as nearly as practicable,
be of uniform weight, age, and parity, and should be representative of
the species and strain under study. Control animals shall be housed, fed,
and handled in a manner identical to that for the test animals, provided,
however, that they shall be caged and housed to preclude or minimize air-
borne or other contamination by the test substance.
(iii) A testing facility shall have a sufficient number of animal
rooms or areas to assure separation of species or test systems and isolation
of individual projects. In addition, there shall be sufficient rooms to
receive, quarantine, and isolate the animals, and to provide for their
routine or (when needed) specialized housing. Structural requirements and
environmental control of these rooms or areas for animals shall comply
with the provisions of the Animal Welfare Act (Pub. L. 94—279) as set
forth in 9 CFR § 3. space requirements for preliminary enclosure shall
also be as specified in 9 CER § 3, except that where specifications regarding
housing of certain species of animals are not set forth, the recommendations
contained in DREW Publication No. (NIH) 78—23 entitled “Guide for the Care
and Use of Laboratory Animals” shall be used. For long term studies,
recommendations of the S publication 1138 are appropriate. See also 40
CFR Part 158 and 40 CFR Part 160.
(iv) Feed and water administered to test animals in chronic studies
shall be chosen so as to minimize contaminant chemical residues. Also,
within a given study, all control animals shall be fed from the same
batch and lot, and shall receive water from the same source, during the
same time period as animals receiving the test substance. If possible,
the feed should be analyzed to assure uniform distribution and adequacy of
nutritional components and combinations with other chemical substances
(e.g., contaminant pesticides, if present).
(6) Caging of test animals . Animals may be group—caged unless a
specific test principle directs otherwise. Minimum space requirements are
outlined in DREW Publication No. (NIH) 78—23. When appropriate, they
should be provided with necessary materials for nesting and shelter. When-
ever signs of morbidity are observed during the test, such affected animals
should be moved to separate cages to avoid cannibalism.
(7) Equipment . All equipment used in conducting the test, including
equipment used to prepare and administer the test substance and equipment
used to maintain environmental conditions, shall be of appropriate design
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and adequate capacity as specified in DHEW Publication No. (NIH) 74—23.
Equipment shall be inspected, cleaned, and maintained regularly. The
equipment shall be properly calibrated.
(8) Observation and clinical testing .
(i) AJ.l observed signs of intoxication and abnormal behavior shall
be recorded at the times of observations throughout the study.
(ii) If a particular kind of clinical test is required to be re-
peated during the test period, the test should be performed on the same
animals whenever possible. However, small animals should not be over—used
for such tests.
(9) Number of animals for tests . The number of animals prescribed in
the principles of the test method for eath section will permit adequate
evaluation of most toxicological effects. If a toxicological effect occurs
with a marginally significant incidence, data from further testing with
larger numbers of animals may be required.
(10) Necropsy procedures . If a section of this subdivision reccxnniends
necropsy examinations be conducted, the following principles should apply:
(i) Procedures to minimize loss of valuable tissues through autolysis
or cannibalism must be employed and should include: undertaking careful
clinical examination of animals to detect those approathing death; killing
and immediately performing necropsy of moribund animals; and isolating
weak animals, to ensure that not nore than 10 percent of the animals are
lost.
(ii) If necropsy cannot be performed immediately after a dead animal
is discovered, the animal should be refrigerated (but not frozen) at tem-
per a tur es 1 ow enough to minimize au to lys is.
(iii) Qualified personnel shall be available so that necropsies can
be performed immediately or as soon as possible, but generally no later
than 16 hours after the time of death.
(iv) Scheduled necropsies shall be performed under the direct super-
vision of a qualified pathologist. [ See paragraph (b)(l)(iii) of this
section.) If histopathology examinations of animal tissues and organs are
also required, the same pathologist should be responsible for both
tasks.
(v) Dead animals and their organs and tissues shall be identified by
reference to the animals’ identification numbers.
(11) Tissue and microslide preparation .
(1) Fixation .
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(A) Tissues and organs destined for microscopic examination should
be placed in 10% buffered formalin or a recognized suitable fixative as
soon as they are removed from the carcass and have undergone necropsy
examination.
(B) Tissues should be fixed no less than 48 hours prior to trimming.
(ii) Trimming . (A) Tissues should be trimmed to a maximum thickness
of 0.4 cm for processing.
(B) Parenchymal organs should be trimmed to allow the largest surface
area possible for microscopic examination. Hollow organs should be trimmed
and blocked to allow a cross section mount to be obtained from mucosa to
serosa. All lymph nodes to be examined should be bisected, preferably
through the hilus.
(C) Tissue trimming should be performed by a pathologist or by a
pathology assistant under the direct supervision of the pathologist.
(See paragraph (b)(1)(iii) of this section.]
(iii) M.icroslide preparation .
(A) Microsections should be routinely 3-5 micrometers thick, and in
no case should a microsection thickness exceed 10 micrometers. All tissues
should be stained with hematoxylin and eosin; however, the use of special
stains appropriate to the individual tissues or lesions is encouraged.
(B) Tissue preparation, block cutting, and slide preparation should
be performed by an HTASCP certified technician or a person having equivalent
training and capability. [ See paragraph (b)(l)(iii) of this section.]
(iv) Identification . Preserved tissues and organs, tissue blocks,
and microscopic slides should be identified by reference to the animals’
identification numbers.
(12) Quality assurance . The testirtg laboratory shall develop and
maintain a system to assure and document adequate performance of its staff
and equipment. This requirement can be met by a Quality Assurance Unit
as described in 40 CFR Part 160 of these guidelines, or by some other
equivalent system of quality control, such as those described in a recent
comprehensive monograph, “Quality Assurance Practices for Health Laboratories”
(Inhorn, 1978). Other references for quality assurance are cited in paragraph
(d) of this section.
(c) Medical or clinical evidence . The Agency may require further
data if medical or clinical evidence or if retrospective epidemiological
evidence suggests that a product or any of its ingredients produces a
toxicological or pharmacological effect not already reflected in the tests
required in this subdivision.
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(d) References . The following are a few examples of publications
thich are available in the rapidly developing field of quality control
(quality assurance):
(1) Bermes, E.W., V. Erviti, and D.T. Forman. 1976. Chapter 2.
Statistics, normal values and quality control. In Fundamentals of Clinical
Q emistry. Tietz, N., ed. W.B. Saunders: Philadelphia.
(2) r aran, M. 1977. Total Quality Control in the Clinical Labora-
tory. C.V. Mosby: St. Louis.
(3) Feigeribaum, A.V. 1961. Total. Quality Control Engineering and
Management. McGraw-Hill: New York.
(4) Galen, R.S., and S.R. Garnbino. 1975. Beyond Normality (The
Predictive Value and Efficiency of Medical Diagnosis). John Wiley and
Sons: New York.
(5) Inhorn, S.L., ed. 1978. Quality Assurance Practices for Health
Laboratories. American Public Health Association: Washington, D.C. 20036.
(6) Re , A.H., and R.J. Henry. 1974. Chapter 12. Accuracy,
precision and control charts. In Clinical Chemistry: Principles and
Technics • 2nd Ed. Henry, R. 3., ed. Harper and Row: New York.
§ 80-4 Reporting of data .
Each test report sulxnitted under this subdivision shall satisfy the
reporting requirements of this section, unless a specific section elsewhere
in this subdivision directs otherwise.
(a) General requirements
(1) Identification . Each test shall identify:
(i) The laboratory where the test was performed, by name and address;
and
(ii) Each party primarily responsible for any written or other matter
contained in the report, and the portions of the report for ‘which he is
responsible.
(2) Verification . Each test report shall be:
(i) Sign by each of the senior scientific personnel, including the
laboratory director, responsible for performing and supervising the testing,
and preparing, reviewing, and approving the test report; and
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(ii) Certified by the applicant or an authorized agent of the applicant
as a canpiete and unaltered copy of the report provided by the testing labora-
tory, whether independent or owned, operated, or controlled by the applicant.
(b) Format and content . The test report shall include all information
necessary to provide a casplete arid accurate description and evaluation of
the test procedures and results. A test report should contain at least three
parts: a summary and evaluation of the test results; a description of the
test procedures; and the data and information required by each applicable
section of this subdivision. Particular information, data, or analysis may
be required sore than once in the test report, and it should be reported or
referenced each time that it is required. Units of measurement must be in
the metric system, but the English system may also be used when appropriate.
In no instance should the systems be mixed (e.g., mg/sq. in.) nor should
both systems be used alternately within a test report.
(1) Summary and evaluation of test results . This section of the test
report shall contain a summary and analysis of the data, and a statement
of the conclusions drawn fran the analysis. The summary should highlight
any and all positive data or observations, and any deviations fran control
data which may be indicative of toxic effects. The summary should be pre-
sented in sufficient detail to permit independent evaluation of the results.
(2) Description of the test procedure . This section of the test
report shall include, but not be limited to, the following information.
If an applicant believes the reporting requirements are inapplicable, he
should subsit an explanatory statement to this effect.
(i) Deviation from standards . The report should indicate all ways in
which the test procedure fails to meet applicable standards for acceptable
testing contained in this subdivision, and should state the reasons for
such deviations. (See 40 CFR Part 158.)
(ii) Methodology . Specification of test methods, including a full
description of the experimental design and procedures, the length of the
study, and the dates on which the study began and ended, shall be stated.
(iii) Substance tested . Identification of the test substance shall
be provided, including:
(A) Chemical name, molecular structure, and a qualitative and quanti-
tative determination of its chemical canposition (including names and
quantities of )c iown contaminants and impurities, so far as is technically
feasible; the determinations should also include quantities of un ciown
materials, if any, so that 100 percent of the sample tested is accounted
for);
(B) Manufacturer and lot number of the test substance; relevant
properties of the substance tested, such as physical state, pH, stability,
and purity; and
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(C) Identification and ccmtposition of any vehicles (e.g., diluents,
suspending agents, and emulsifiers) or other materials used in administering
the test substance.
(iv) Animal data . Animal data shall include:
(A) Species and strain used, rationale for selection of species (if
the species is other than the species preferred or required by sections of
this subdivision), and rationale for selection of strain;
(B) Source of supply of the animals;
(C) Description of any pre—test conditioning, including diet and
quarantine;
(D) Method of randcznization used in assigning animals to test or
control groups;
CE) Numbers of animals of each sex in each test or control group and
(F) Age and condition of animals at beginning of study.
Cv) Environmental conditions . A description of the caging condi-
tions shall include: number (and any thange in number) of animals per
cage, bedding material, ambient temperature and humidity, photoperiod,
and identification of the diet of the test animal.
(vi) Dosing . Dosing information s hall include:
(A) All dose levels administered;
(B) Method and frequency of administration (including hour of dosing in
relation to photoperiod);
(C) Total volume of material (i.e., test substance plus vehicle)
individual dosings;
CD) Duration of treatment;
CE) If the test substance is administered in the feed or by another
vehicle, the method of randanization used in selecting samples to assay,
the assay method used to determine the stability and homogeneity of the
test substance being administered, and the results of this assay;
(vii) Treatment for infectious diseases . A description of the treat-
ment(s) used to prevent or control infectious diseases if suth treatment
was undertaken during a test or shortly before a test was begun. Such a
description shall include, for eath individual affected animal:
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(A) Its identification number;
(B) The nature and severity of the disease, if present;
(C) The date of first observation and duration of disease, if present;
(D) The nature of the treatment for disease or disease prevention, and
the dates of suth treatment; and
(E) The outcane of the treatments in relation to the disease and to the
test results.
(viii) Observations . Frequency, duration, and method of observation of
the animals.
(ix) Availability of raw data, specimens, and.samples of the test sub-
stances . The location of all raw data, specimens, and samples of the test
substances which are retained in accordance with 40 CFR Part 160 and the
name and address of the individual who is responsible for the ardiives.
(x) References . Statistical and any other methods employed for analyz-
ing the raw data, a list of references to any published literature used in
developing the test protocol, performing the testing, making and interpreting
observations, and canpiling and evaluating the results.
(3) Reporting requirements for specific tests . This section of the
test report should include all data, information, and analysis required by the
“Data r orting and evaluation” paragraphs of the sections in this subdivision.
(c) Statistical procedures .
(1) General . Statistical techniques are required for several toxicologi-
cal analyses, such as the LD5O calculations for acute oral and acute dermal
toxicity studies ( 81—1 and 81—2), the LC5O calculations for acute inhalation
toxicity study ( 81—3), and the median particle size analyses used to describe
the aerosol clouds in the acute and subthronic inhalation studies ( 81-3 and
82-4). Median lethal doses are to be measured within a 95% confidence limit
of 20% of their median, then technically feasible. When not feasible, e.g.,
due to inherently variable responses or to difficulties in administering the
test substance, the registration applicant should explain why the limit was
exceeded. In addition, appropriate statistical methods shall be used to
stunniarize experimental data, to express trends, and to evaluate the signifi-
cance of differences in data from individual test groups. The methods used
shall reflect the current state of the art. A list of references in paragraph
(d) of this section represents some of the techniques currently in use.
(2) Standard deviation and standard error . All data averages or means
shall be acccxnpanied by standard deviation, to indicate the amount of variability
in the raw data. In addition, the standard errors of the means may also be
calculated, since they are useful in canparing means fran different test groups;
however, notations of statistically significant differences, accanpanied by the
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confidence level or probability, may be used in place of the standard errors.
Other methods of expressing data dis rs ion may also be used, sthen appropriate.
(d) References .
(1) The following are a few of many good textbooks in statistics:
Ci) Remington, R.D., and M.A. Schork. 1970. Statistics with Applica-
tions to the Biological and Health Sciences. Prentice-Hall: New York. [ In-
cludes a thapter on non—parametric methods (chapter 12. Distribution Free
and Nonparametric Methbds) which are useful for non—nonnally distributed
data].
(ii) Rohif, F.J., and R.R. Sokal. 1969. Statistical Tables. W.H. Freeman
and Company: San Francisco.
(iii) Sokal, R.R., and F.J. Rcthlf. 1969. Biometry. W.H. Freeman and
Company: San Francisco.
(iv) Von Fraunhofer, J.A., and J.J. rray. 1976. Statistics in
Medical, Dental and Biological Studies. Thi—Med Books Limited:
London.
(2) The following are examples of available canputer programs ich
can be used in the statistical processing of data but generally involve a
large computer operation. There are also many desk-top minicomputers which
supply similar computer programs for statistical analyses.
(i) Dixon, W.J., ed. 1970. Biomedical Computer Programs (BMD). 2nd
Ed. University of California Press: Los Angeles.
(ii) Nie, N.H., C.H. Hull, J.G. Jenkins, K. Steinbrenner, and D.H. Bent.
1975. Statistical Package for the Social Sciences (SPSS). 2nd Ed.
McGraw—Hill: New York.
§ 80—5 Combined testing .
(a) Policy . In order to encourage efficient use of test animals and
laboratory resources, the data required by this subdivision may be derived
from test methodologies which satisfy the principles for acceptable testing
contained in two or riore different sections of this subdivision.
(b) Procedures . (1) Principles of Test Methods . Where combined
testing is conducted, the standards for acceptable testing contained in this
subdivision for each of the data requirements should be satisfied or, in
accordance with Part 158, Subpart B, an applicant should establish that the
purposes of the standards would be satisfied by the combined testing protocol.
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(2) Consultation . Prior to initiating a combined test methodology, an
applicant is encouraged to consult with the Agency to determine whether the
proposed combined test methodology would be acceptable.
(c) Combined testing instruction in test sections . Several sections
of this subdivision detailing specific test requirements provide specific
instruction on combined testing procedures. See especially 83—5, a
section devoted entirely to the combined chronic toxicity — oncogenicity
testing.
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Series 81: ACUTE TOXICITY ND IRRITATION STUDIES
(NOTE: The sections of this series are prepared in conformity with the
guidelines developed by the Organization of Economic Cooperation and Develop-
ment.]
§ 81—1 Acute oral toxicity study .
(a) When req uired . Data on the single-dose oral LD5O are required
by 40 CFR Part 158 to support the registration of each manufacturing-use
product and each end—use product, unless the substance to be tested under
paragraph (e) of this section is a gas or highly volatile substance that
cannot be administered orally. See, specifically, 40 CFR § 158.50 and §
158.135 to determine whether these data must be submitted. Section Il—A
of this subdivision contains an additional discussion of the “Formulators’
Exemption” and who, as or general rule, is responsible for submission of
the required data.
(b) Purpose . In the assessment and evaluation of the toxic character-
istics of a substance, determination of acute oral toxicity is usually an
initial step. It provides information on health hazards likely to arise
from a single exposure by the oral route. The purpose of the acute oral
study is to determine the median lethal dose (LD 50), its statistical
limits and slope using a single exposure and a 14—day post—exposure observa-
tion period. Data from an acute study serves as a basis for classification,
labeling, and packaging. It is also an initial step in establishing a
dosage regimen in subchronic and other studies and may provide initial
information on the mode of toxic action of a substance.
(c) Definitions . (1) “Acute oral toxicity” is the adverse effects
occurring from the oral administration of a single dose of a substance.
(2) “Dosage” is a general term comprising the dose, its frequency and
the duration of dosing.
(3) “Dose” is the amount of test substance administered. Dose is
expressed as weight of test substance (g, mg) per unit weight of test animal
(e.g., mg/kg).
(4) “Dose-effect” is the relationship between the dose and the
magnitude of a defined biological effect e her in an individual or in a
population sample.
(5) “Dose—response” is the relationship between the dose and the
proportion of a population sample showing a defined effect.
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(6) “LD5O (median lethal dose), oral” is a statistically derived
single dose of a substance that can be expected to cause death in 50 percent
of animals when administered by the oral route. The LD5O value is expressed
in terms of weight of test substance (g, mg) per unit weight of test animal
(e.g., mg/kg).
(d) Principle of the test method . The test substance is administered
orally by gavac in graduated doses to several groups of experimental ani-
mals, one dose being used per group. Subsequent observations of effects
and deaths are made. Animals which die during the test are necropsied, and
at the conclusion of the test the surviving animals are sacrificed and
necropsied as indicated by the nature of the toxic effects observed. This
principle is directed primarily to studies in rodent species but may be
adapted for studies in nonrodents.
(e) Substance to be tested . (1) The manufacturing—use product and,
if different, the technical grade of each active ingredient shall be tested
to support the registration of a manufacturing—use product.
(2) The end—use product shall be tested to support the registration
of an end—use product.
(3) The end—use product, as diluted for use in accordance with labeling
directions, shall be tested to support the registration of each end—use
product intended for domestic application.
(4) If the toxicity of the use dilution or of the end—use product can
be established from tests performed on other use dilutions or on other end-
use products for which registration is sought, the use dilution need not be
separately tested.
(f) Limit test . If a test at one dose level of at least 5000 mg/kg
body weight, using the procedures described for the study, produces no
compound—related mortality, then a full study using a minimum of three
dose levels might not be necessary.
(g) Test procedures . (1) Animal selection . (i) Species and strain .
Although several mammalian test species may be used, the rat is the preferred
rodent species. Commonly used laboratory strains should be employed. If
another species is used, the tester should provide justification/reasoning
for its selection.
(ii) . Young adult animals should be used. The weight variation
of animals used in a test should not exceed ± 20 percent of the mean weight
for each sex.
(iii) Sex. (A) Equal numbers of animals of each sex are required
for each dose level.
(B) The females should be nulli.parous and non-pregnant.
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(iv) Numbers . At least 10 animals (5 female and 5 male) at each dose
level should be used.
(2) Control groups . Neither a concurrent untreated nor vehicle con-
trol group is recanmended except when the toxicity of the vehicle is unknown.
(3) Dosing . (1) Dose levels and dose selection . At least three
dose levels should be used and spaced appropriately to produce test groups
with a range of toxic effects and n rta].ity rates. The data should be
sufficient to produce a dose response curve and, where possible, permit an
acceptable determination of the LD5O.
(ii) Vehicle . Where necessary, the test substance is dissolved or
suspended in a suitable vehicle. It is recanmended that wherever possible
the usage of an aqueous solution be considered first, followed by consid-
eration of a solution in oil (e.g., corn oil) and then by possible solu-
tion in other vehicles. For non—aqueous vehicles the toxic tharacteristics
of the vehicle should be known, and if not known should be determined
before the test.
(iii) Volume . The maximum volume of liquid that can be administered
at one time depends on the size of the test animal. In rodents, the volume
should not exceed 1 ml/100 g body weight, except in the cases of aqueous
solutions where 2 ml/100 g may be used. Variability in test volume should
be minimized by adjusting the concentration to ensure a constant volume at
all dose levels.
(4) Observation period . The observation period should be for at
least 14 days. However, the duration of observation should not be fixed
rigidly. It should be determined by the toxic reactions, rate of onset and
length of recovery period, and may thus be extended when considered neces-
sary. The time at which signs of toxicity appear and disappear, their
duration and the time to death are important, especially if there is a
tendency for deaths to be delayed.
(5) Exposure . Ci) The test substance should be administered in a
single dose by gavage, using a stanath tube or suitable intubation cannula.
(ii) Animals should be fasted prior to substance administration. For
the rat, food should be withheld overnight; for other rodents with higher
metabolic rates a shorter period of fasting is appropriate.
(iii) After the substance has been administered, food may be withheld
for a further 3—4 hours.
(iv) If a single dose is not possible, the dose may be given in smaller
fractions over a period not exceeding 24 hours. Where a dose is administered
in fractions over a period, it may be necessary to provide the animals with
food and water, depending on the length of the period.
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(6) Observation of animals .
(1) A careful clinical camination should be made at least once
each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation of weak or moribund
animals.
(iii) Cageside observations should include, but not be limited to,
thanges in:
(A) The skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Autonemic and central nervous system;
(F) Somatornobor activity; and
(G) Behavior pattern.
(H) Particular attention should be directed to observation of tremors,
convulsions, salivation, diarrhea, lethargy, sleep and coma.
(iv) Individual weights of animals should be determined shortly before
the test substance is administered, weekly thereafter and at death. Changes
in weight should be calculated and recorded when survival cceeds one day.
(v) The time of death should be recoraed as precisely as possible.
(vi) At the end of the test surviving animals are weighed and
sacrificed.
(7) Gross pathology . Consideration should be given to performing a
gross necropsy of all animals where indicated by the nature of the toxic
effects observed. All gross pathological thanges should be recorded.
(h) Data and reporting . (1) Treatment of results . Data shall be
si.munarized in tabular form, showing, for each test group:
(i) The number of animals at the start of the test;
(ii) Time of death of individual animals at different dose levels;
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(iii) Number of animals displaying other signs of toxicity;
(iv) Description of toxic effects; and
(v) Necropsy findings.
(2) Evaluation of results . The LD5O value shall always be considered
in conjunction with the observed toxic effects and any necropsy findings.
The LD5O value is a relatively coarse measurement useful only as a reference
value for classification, labeling purposes, and packaging, for expressing the
possible lethal potential of the test substance by the ingestion route.
Reference should always be made to the experimental animal species in sthich
the LD5O value was obtained. An evaluation should include the relationship,
if any, between the animals exposed to the test substance and the incidence
and severity of all abnormalities, including:
(i) Behavioral abnormalities;
(ii) Clinical abnormalities;
(iii) Gross lesions;
(iv) Body weight dianges;
(v) Effects on mortality; and
(vi) Any other toxicological effects.
(3) Test report . Iii addition to the information recanmended by 80-4,
the test r ort summary shall include the following information:
(i) Tabulation of response data by sex and dose level (i.e., number
of animals dying; number of animals showing signs of toxicity; number of
animals exposed);
(ii) Description of toxic effects;
(iii) Time of death after dosing;
(iv) LD5O value for each sex determined at 14 days (with the
method of determination specified);
(v) Ninety—five percent confidence interval for the LD5O;
(vi) Dose—mortality curve and slope ( there permitted, by the
n thod of determination);
(vii) Body weights when taken;
(viii) Necropsy findings
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(ix) Pathology findings, when performed.
§ 81-2 Acute dermal toxicity study .
(a) When rep.iired .
(1) Routine testing . Data on the single-dose dermal LD5O are required
by 40 CFR Part 158 to support the registration of each manufacturing—use product
and each end—use product, unless the substance which would be tested under
paragraph (e) of this section is corrosive or a gas or highly volatile
substance that cannot be administered dermally.
(2) Use dilution testing . Data from tests performed with the use
dilutions of a product may be required if the use dilution is intended for
non—domestic application as a mist or spray. Applicants should consult with
the Agency to determine the principles for such testing, if required.
(3) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
Whether these data must be submitted. Section 11—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must submit the required data as a general rule.
(b) Purpose . In the assessment and evaluation of the toxic diaracter-
istics of a substance, determination of acute derrnal toxicity is useful
where exposure by the dermal route is likely. The purpose of the acute
dermal study is to determine the median lethal dose (LD5O), its statistical
limits and slope using a single exposure over a 24-hour period and a 14-day
post-exposure observation period. Data fran an acute dermal toxicity study
serve as a basis for classification, labellng, and packaging. It is also
an initial step in establishing a dosage regimen in subthronic and other
studies and may provide info rma t ion on de rmal abso rpt ion and the mode of
toxic action of a substance by this route.
(C) Definitions .
(1) “Acute dermal toxicity” is the adverse effect occurring during or
following a 24—hours dermal exposure to a single dose of a test substance.
(2) “Dosage” is a general term comprising the dose, its frequency and
the duration of dosing.
(3) “Dose” is the amount of test substance applied. Does is expressed
as weight of test si bstance (g, mg) per unit weight of test animal (e.g., mg/kg).
(4) “Dose—effect” is the relationship between the dose and the
magnitude of a defined biological effect either in an individual or in a
population sample.
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(5) “Dose-response” is the relationship between the dose and the
proportion of a population sample showing a defined effect.
(6) “LDSO (median lethal dose) dermal” is a statistically derived
single dose of a substance that can be expected to cause death in 50
percent of t eated animals when applied to the skin. The LD5O value is
expressed in terms of weight of test substance (g, mg) per unit weight of
test animal (e.g., mg/kg).
(d) Principle of the test method . The test substance is applied to
the skin in graduated doses to several groups of experimental animals, one
dose being used per group. Subsequently, observations of effects and deaths
are made. Animals which die during the test are necropsied, and at the
conclusion of the test the surviving animals are sacrificed and necropsied
as indicated by the nature of the toxic effects observed.
(e) Substance to be tested .
(1) The manufacturing—use product and, if different, the technical
grade of each active ingredient shall be tested to support the registration
of a manufacturing—use product.
(2) The end—use product shall be tested to support the registration
of an end—use product.
(3) If the toxicity of the end-use product can be established fran
tests performed on other end-use products, the end—use product for which
registration is sought need not be separately tested.
(f) Limit test . If a test at a dose of at least 2000 mg/kg body
weight, using the procedures described for this study, produces no canpound—
related mortality, then a full study using a minimum of three dose levels
might not be necessary.
(g) Test procedures . (1) Animal selection . (i) Species and
strain . The rat, rabbit or guinea pig may be used. The albino rabbit is
preferred because of its size, ease of handling, skin permeability and
extensive data base. Caninonly used laboratory strains should be employed.
If a species other than the three indicated above is used, the tester
should provide justification/reasoning for its selection.
(ii) . Adult animals should be used. The following weight ranges
are suggested to provide animals of a size which facilitates the conduct
of the test: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350
to 450 g.
(iii) Sex. (A) Equal numbers of animals of each sex with healthy
intact skin are recamnended for each dose level.
(B) The females should be nulliparous and non—pregnant.
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41
(iv) Numbers . At least 10 animals (5 females and 5 males) at each dose
level should be used.
(2) Control groups . Neither a concurrent untreated nor vehicle control
group is recanmended except when the toxicity of the vehicle is unknown.
(3) Dosing . (1) Dose levels and dose selection . At least three
dose levels should be used and spaced appropriately to produce test groups
wi th a range of toxic effects and u rtal I ty rates • The da ta should be
sufficient to produce a dose—response curve and, where possible, permit an
acceptable determination of the LD5O.
(ii) Vehicle . Where necessary, the test substance is dissolved or
suspended in a suitable vehicle. It is reccinmended that wherever possible
the usage of an aqueous solution be considered first, followed by considera-
tion of a solution in oil (e.g., corn oil) and then by possible solution
in other vehicles. For non—aqueous vehicles the toxic tharacteristics of
the vehicle should be known, and if not known should be determined before
the test.
(4) Exposure duration . The duration of exposure should be approxi-
mately 24 hours.
(5) Observation period . The observation period should be at least 14
days. However, the duration of observation should not be fixed rigidly. It
should be determined by the toxic reactions, rate of onset and length of
recovery period, and may thus be extended when considered necessary. The
time at which signs of toxicity appear and disappear, their duration and the
time of death are important, especially if there is a tendency for deaths to
be delayed.
(6) Preparation of animal skin . (i) Approximately 24 hours before
the test, fur should be removed from the dorsal and ventral area of the
trunk of the test animals by clipping or shaving. Care must be taken to
avoid abrading the skin which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be
clear for the application of the test substance • The weight of the animal
should be taken into account when deciding on the area to be cleared and
on the dimensions of the covering.
(iii) When testing solids, which may be pulverized if appropriate,
the test substance should be moistened sufficiently with water or, where
necessary, a suitable vehicle to ensure good contact with the skin. When
a vehicle is used, the influence of the vehicle on penetration of skin by
the test substance should be taken into account.
(7) Application of test substance . (I) The test substance should
be applied uniformly over an area which is approximately 10 percent of the
total body surface area. With highly toxic substances the surface area
co iered may be less, but as much of the area should be co’,ered with as
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thin and uniform a film as possible. In the case where less than 10% of
the surface area is covered an approximation of the exposed areas shou.ld
be determined.
(ii) Test substance should be held in contact with the skin with a
porous gauze dressing and non—irritating tape throughout a 24—hour exposure
period. The test site should be further covered in a suitable manner to
retain the gauze dressing and test substance and ensure that the animals
cannot ingest the test substance. Restrainers may be used to prevent the
ingestion of the test substance, but c nplete imn bilization is not a
recanmended method.
(iii) At the end of the exposure period, residual test substance should
be removed, where practicable using water or an appropriate solvent.
(8) Observation of animals . (i) A careful clinical examination
should be made at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation of weak or moribund
animals.
(iii) Cageside observations should include, but not be limited to,
d anges in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Autonanic and central nervous system;
(F) Somatomotor activity; and
(G) Behavior pattern.
(H) Particular attention should be directed to observations of tremors,
convulsions, salivation, lethargy, sleep and coma.
(iv) Individual weights of animals should be determined shortly before
the test substance is applied, weekly th3reafter, and at death. Changes
in weight should be calculated and recorded when survival exceeds one day.
(v) The time of death should be recorded as precisely as possible.
(vi) At the end of the test, surviving animals are weighed and
sacrificed.
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(9) Gross pathology . Consideration should be given to performing a
gross necropsy of all animals Inere indicated by the nature of the toxic
effects observed. All gross pathological dianges should be recorded.
(h) Data and reporting .
(1) Treatment of rpsults . Data shall be summarized in tabular
form, showing, for eadi test group:
(i) The number of animals at the start of the test;
(ii) Time of death of individual animals at different dose levels;
(iii) Number of animals displaying other signs of toxicity;
(iv) Description of toxic effects; and
(v) Necropsy findings.
(2) Evaluation of results . The dermal LD5O value shall always be
considered in conjuction with the observed toxic effects and the necropsy
findings. The LD5O value is a relatively coarse measurenent, useful as
a reference value for classification and labeling purposes, packaging and
expressing the possible lethal potential of the test substance following
dermal exposure. Reference should always be made to the experimental
animal species in which the LD5O value was obtained. An evaluation should
include the relationship, if any, between the animals exposed to the
the test substance and the incidence and severity of all abnormalities,
including:
(i) Behavioral abnormalities;
(ii) Clinical abnormalities;
(iii) Gross lesions;
(iv) Body weight thanges;
(v) Effects on mortality; and
(vi) Any other toxicological effects.
(3) Test report . In addition to the information r uired by § 80—4,
the test report summary shall include the following information:
(i) Tabulation of response data by sex and dose level (i.e., number
of animals dying, number of animals showing signs of toxicity, number of
animals exposed);
(ii) Description of toxic effects;
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(iii) Time of death after dosing;
(iv) LD5O value for each sex de termined at 14 days (wi th the method
of determination specified);
(v) Ninety—five percent confidence interval for the LD5O;
(vi) Dose—mortality curve and slope ( ithere permitted b the method of
determination);
(vii) Body weLghts when taken; arid
(viii) Pathology findings, when performed.
(ix) The approximate amount of material applied per unit of
skin exposed (calculated in mg per square n of skin).
§ 81—3 Acute inhalation toxicity study .
(a) ‘then required .
(1) A determination of the acute inhalation toxicitY is required by
40 CPR Part 158 to support the registration of a manufactu± .n USe product, if:
(i) The product is a gas;
(ii) The product is a solid or a liquid which may produce a signifi-
cant vapor hazard based on its toxicity and expected use; or
(iii) The product contains particles of inhalable size for man (that
is, it contains particles of aerodynamic diameters of 15 micrometers or
less).
(2) A determination of the acute inhalation toxicity is required
to support the registration of an end-use product, if;
(i) The end-use product (as registered or under conditions of
use) is a gas;
(ii) The product is a solid or a liquid which may pro&ce a signif i-
cant vapor hazard based on its toxicity arid expected use; or
(iii) The product under conditions of use will produce inha].able liquid
or solid particles (that is, particles of aerodynamic diameter of 15 micrometers
or less).
FReproduced from
best available copy.
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(iv) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be submitted. Section 11-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
ho must submit the required data as a general rule.
(b) Purpose . In the assessment and evaluation of the toxic dtarac—
teristics of an irihalable material, such as a gas, volatile substance or
aerosol/particulate, determination of acute inhalation toxicity is an
initial step. It provides information on health hazards likely to arise
from short term exposure by the inhalation route. The purpose of the
acute inhalation study is to determine the median lethal concentration
(LC5O), its statistical limits and slope using a single exposure, usually
of 4 hours, and a 14-day poet-exposure observation period. Data fran an
acute study serves as a basis for classification and labeling. It is
also an initial step in establishing a dosage regimen in subthronic and
other studies and may provide additional information on the mode of toxic
action of a substance.
Cc) Definitions .
(1) “Acute inhalation toxicity” is the total adverse effects caused
by a substance following a single uninterrupted exposure by inhalation
over a short period of time to a subs tance capable of being inhaled.
(2) “Aerodynamic diameter” applies to the behavioral size of
particles of aerosols. It is in diameter of a sphere of unit density which
behaves aerodynamically as the particle of the test substance. It is used
to canpare particles of different sizes, shapes and densities and to
predict where in the respiratory tract such particles may be deposited.
This term is used in contrast to “optical,” “measured” or “gecme tr “
diane te rs which are representation of actual diane ters which in themselves
cannot be rela ted to deposition within the respiratory tract.
(3) “Gecme tric mean diameter” or “median diane ter” is the calculated
aerodynamic diameter which divides the particles of an aerosol in half
based on the weight of the particles. Fifty percent of the particles by
weight will be larger than the median diameter and 50 percent of the
particles will be smaller than the median diameter. The median diameter
and its gecinetzic standard deviation are used to statistically describe the
particle size distribution of any aerosol based on the weight and size of
the particles.
(4) “Inhalab le diameter” refers to that aerodynamic di ame ter of a
particle which is considered to be inhalable for the organism. It is used
to refer to particles which are capable of being inhaled and may be depos-
ited anywhere within the respira tory tract from the trachea to the deep
lung (the aveoli) • For Dan, the inhalable diameter is considered here as
15 microseters or less.
(5) “LC50” (median lethal concentration) is a statistically derived
concentration of a substance that can be expected to cause death during
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exposure or within a fixed time after exposure in 50 percent of animals
exposed for a specified time. The LC5O value is expressed as weight or
volume of test substance per volume of air (e.g., mg/i or ppm).
(d) Principle of the test method . Several groups of experimental
animals are to be exposed for 4 hours to the test substance in graduated
concentrations, one concentration being used per group. Where a vehicle
is required, it ideally should not substantially alter the themical or
toxicological properties of the test substance. Subsequent observations
of effects and deaths are made. Animals thich die during the tests are
necropsied and at the conclusion of the test surviving animals are
sacrificed and necropsied as necessary.
Ce) Substance to be tested . (1) The manufacturing—use product and,
if different, the technical grade of eadi active ingredient, shall be
tested to support the registration of a manufacturing—use product.
(2) The end-use product shall be tested to support the registration
of an end-use product.
(3) The diemical ccinposition and physical state of the substance
being tested should, if possible, be the same as that thicth is encountered
during the use of the product. Aerosol particles may have to be reduced
to sizes whith are inhalable for the animal being tested considering the
entire respiratory system of the animal.
(f) Limit test . If a test at an exposure of 5 mg/l (actual con-
centration of respirable substances) for 4 hours or, where this is not
possible due to physical or themical properties of the test substance, the
maximum attainable concentr tion, produces no canpound-rela ted n rbal i ty,
then a full study using three dose levels might not be neCessary.
(g) Test procedures . (1) Animal selection . Ci) Species and
strain . Although several mammalian test species may be used, the preferred
species is the rat. Commonly used laboratory strains should be used. If
another mammalian species is employed, the tester should provide justifica-
tion/reasoning for its selection.
(ii) Young adult animals should be used. The weight variation of
animals or between groups of animals used in a test should not exceed +
20 percent of the mean weight for each sex.
(iii) Sex. (A) Equal numbers of animals of eath sex are recommended for
each dose level.
(B) The females should be nulliparous and non-pregnant.
(iv) Numbers . At least 10 animals (5 female and 5 male) at each dose
level should be used (except if the limit test applies).
(2) Control groups . Neither a concurrent untreated nor vehicle con-
trol group is required except when the toxicity of the vehicle is unknown.
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(3) Dose levels and dose selection . (i) At least three exposure
concentrations should be used and spaced appropriately (between the 10%
and 90% mortality range) to produce test groups with a range of toxic
effects and mortality to produce a concentration mortality curve and permit
an aoceptable determination of an LC50. One dose level with 0% and/or
100% mortalities adjacent to the 10% to 90% dose level may be used to
calculate the LC50.
(ii) Where necessary, a suitable vehicle may be added to the test
substance to help generate an appropriate concentration of the test substance
in the atmosphere. If a vehicle or diluent is needed, ideally it should
not elicit important toxic effects itself nor substantially alter the
chemical or toxicological properties of the test substance.
(iii) In the case of potentially explosive test substances, care
should be taken to avoid generating explosive concentrations.
(4) Exposure duration . The duration of exposure should be at least
four hours allowing appropriate additional time for chamber equilibrium.
(5) Observation period . The observation period should be at least
14 days. However, the duration of observation should not be fixed rigidly.
It should be determined by the toxic reactions, rate of onset and length of
recovery period, and may thus be ctended when considered necessary. The
time at which signs of toxicity appear and disappear, their duration and
the time of death are important, especially if there is a tendency for
deaths to be delayed.
(6) Inhalation exposure . (i) The animals should be tested with
inhalation equipment designed to sustain a dynamic air flow of 10 air
changes per hour, ensure an adequate oxygen content of at least 19 percent
and an evenly dis tributed exposure atmosphere. Where a chamber is used
its design should minimize crowding of the test animals and maximize their
exposure to the test substance. As a general rule to ensure stability of
a chamber atmosphere, the total “volume” of the test animals should not
exceed 5 percent of the volume of the test chamber. Maintenance of a
slight negative pressure inside the chamber will prevent leakage of the
test substance into surrounding area.
(ii) The temperature at which the test is performed should be main-
tained at 22°C (+ 2°) for the rat. The relative humidity should be main-
tained between 40 to 60 percent unless the nature of the test substance
or generating procedure (such as using water as a vehicle) precludes this.
(iii) Alternatively, oro—nasal, or head only exposures may be used
if the animals exposed in chambers are excessively coated with test substance
and/or the whole body exposures produce high toxicity in the face or low
oral or derntal toxicity.
(7) Physical measurements . Measurements and/or monitoring should be
made of the following:
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(1) The rate of air flow should be recorded at least every 30 minutes.
Electronic nitoring of flow is desirable.
(ii) Actual concentrations of the test substance of the atmosphere
from the breathing zone of the animals should be determined. Samples
should be taken of ten enough to adequa tely characterize the atmosph es to
hith the animals are exposed (at least twice during the exposure, one
after initial diamber equilibration and one late in the exposure).
(iii) For exposures to aerosols, aerodynamic particle size analyses
should be performed to establish the distribution of the sizes of the
particles and the consistancy of the aerosol generating system. Particle
size analyses during the animals’ test exposures should then be carried
out as often as necessary to tharacterize the aerosols to ith the animals
are exposed.
(iv) Temperature should be recorded at least every 30 minutes.
Electron onitoring of temperature is desirable.
(8) Food and water during exposure period . Food should be withheld
during exposure and water should not come in direct contact with the
test atmospheres.
(9) Observation of animals . (i) The animals should be observed
clinically at least daily and actions taken to minimize loss of animals to
the study, e.g., necropsy or refrigeration of those animals found dead and
isolation of weak or nrribund animals.
(ii) Cageside observations should include, but not be ilmited to,
thanges in:
(A) The skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Auto nomic and central nervous sys tern;
(F) Somatomotor activity; and
(G) Behavior pattern.
(H) Particular attention should be directed to observation of tremors,
convulsions, salivation, diarrhea, lethargy, sleep and coma.
(iii) Individual weights of animals should be determined shortly before
the test substance is administered, weekly thereafter, and at death. Changes
in weight should be calculated and recorded when survival exceeds one day.
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(iv) The time of death should be recorded as precisely as possible.
Cv) At the end of the test the surviving animals are weighed and
sacrificed.
(10) Gross pathology . Consideration should be given to performing
a gross necropsy of all animals where indicated by the nature of the toxic
effect observed with particular reference to any changes in the respiratory
tract. Where there are significant signs of toxicity indicating the pos-
sible involvement of other organs these should be examined and all gross
pathological thanges recorded.
(11) Histopathology . Microscopic examination of organs showing
evidence of gross pathology in animals surviving 24 hours or core shall be
considered since it may yield useful information.
(h) Data and reporting . (1) Treatment of results . Data should
be summarized in tabular form, showing, for each test group:
(i) The number of animals at the start of the test;
(ii) Time of death of individual animals at different exposure levels;
(iii) Number of animals displaying other signs of toxicity;
(iv) Description of toxic effects; and
(v) Necropsy findings.
(2) Evaluation of results . The LC50 value shall always be considered
in conjunction with the observed toxic effects and the necropsy findings. The
LC5O value is a relatively coarse measurement, useful only as a reference
value for classification and labeling purposes, and expressing possible
lethal potential of the test substance following inhalation. Reference
should always be made to the experimental animal species in whith the LC50
value was obtained. An evaluation should include the relationship, if any,
between the animals’ exposure to the test substance and the incidence and
severity of all abnormalities, including:
(i) Behavioral abnormalities;
(ii) Clinical abnormalities;
(iii) Gross lesions;
(iv) Body weight anges;
Cv) Mortality; and
(vi) Other toxicological effects.
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(3) Test report . In addition to the information required by
§ 80—4, the test report summary shall include the following information:
(i) Test conditions . (A) Description of exposure apparatus includ-
ing design, type, dimensions, source of air, system for generating particu—
lates and aerosols, methods of conditioning air, and the method of housing
the animals in a test thamber when this apparatus is used.
(B) The equipment for measuring temperature, humidity, and particulate
aerosol concentrations and size shall be described.
(ii) Exposure data . These shall be tabulated and presented with
mean values and a measure of variability (e.g., standard deviation) and
should include:
(A) Airflow rates through the inhalation equipment;
(B) Temperature of air;
(C) Nominal concentration——total amount of test substance fed into
the inhalation equipment divided by volume of air (no standard deviation);
(D) Measured total concentrations (particulate and/or gaseous phases)
in test breathing zone; and
(E) Particle size distribution (e.g., median aerodynamic diameter of
particles with geometric standard deviation) including estimates of the
percents of inh alab le and non—inh al able portions for the tes t animals.
(iii) Animal data . (A) Tabulation of response data by sex and
exposure level (i.e., number of animals dying, number of animals showing
signs of toxicity, number of animals exposed, species and strain used);
(B) Description of toxic effects;
(C) Time of death during or following exposure;
(D) LC5O for each sex determined at 14 days (with method of calcula-
tions specified):
(E) Ninety—five percent confidence interval for the LC50;
(F) Dose—nortality curve and slope (where permitted by the method
of determination);
(G) Body weights, when taken; and
(H) Pathology findings, when performed.
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§ 81—4 Primary eye irritation .
(a) When required . Data on primary eye irritation are required by
40 CFR Part 158 support the registration of each manufacturing-use
product and each end—use product. See, specifically, 40 CFR § 158.50 and
§ 158.135 to determine whether these data must be subsitted. Section 11-A
of this subdivision contains an additional discussion of the “Formulators’
Exemption” and who must subsit the required data as a general rule.
(b) Purpose . In the assessment and evaluation of the toxic diaracter—
istics of a substance, determination of the irritant and/or corrosive effects
on eyes of mammals is an important initial step. Information derived fran
this test serves to indicate the existence of possible hazards likely to
arise fran exposure of the eyes and associated muc s membranes to the test
substance.
(c) Definitions .
(1) “Eye corrosion” is the production of irreversible tissue damage
in the eye following application of a test substance to the anterior surface
of the eye.
(2) “Eye irritation” is the production of reversible thanges in the
eye following the application of a test substance to the anterior surface
of the eye.
(a) Standard of the test method .
(1) The substance to be tested is applied in a single dose to one of
the eyes in e a di of several experimental an ima is; the un trea ted eye is
used to provide control information. The degree of irritation/corrosion
is evaluated and scored at specific intervals and is fully described to
provide a ccmiplete evaluation of the effects. The duration of the study
should be sufficient to permit a full evaluation of the reversibility or
irreversibility of the effects observed but need not exceed 21 days.
(2) Strongly acidic or alkaline substances, for example with a derron—
strated pH of 2 or less or 11.5 or greater, need not be tested owing to
their predictable corrosive properties.
(3) Materials which have demonstrated definite corrosion or severe
irritation in a dermal study need not be further tested for eye irritation.
It may be presumed that such substances should produce similarly severe
effects in the eyes. -
(e) Substance to be tested . (1) Test substance . (i) The manufac-
turing-use product shall be tested to support the registration of a
manufacturing—use product.
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(ii) The end—use product shall be tested to support the registration
of an end—use product.
(2) Condition of test substance .
(i) If the test substance is a liquid, it should be placed in the
eye undiluted, in accordance with paragraph (f)(4) of this section.
(ii) If the test substance is a solid or granular product, it should
be ground into a fine dust or powder. The test substance should not be
moistened before it is placed in the eye in accordance with paragraph
(f)(4) of this section.
(3) Corrosive pesticides . Data which demonstrate that the test sub-
stance specified by paragraph (e)(1) of this section has a pH of 1—2 or
11.5—14 may be submitted in lieu of data fran a primary eye irritation study
conducted in accordance with paragraph (f) of this section. For all regula-
tory purposes, the Agency will assume that such a substance is corrosive.
(f) Test procedures . (1) Animal selection . (i) Species and
strain . A variety of experimental animals have been used, but it is recan-
mended that testing should be performed using healthy adult albino rabbits.
Commonly used laboratory strains should be used. If another mammalian
species is used, the tester should provide justification/reasoning for
its selection.
(ii) Number of animals . At least six animals should be used, unless
justification/reasoning for using fewer animals is provided.
(2) Dose level . Ci) For testing liquids, a dose of 0.1 ml is
used. In testing solids, pastes, and particulate substances, the amount
used should have a volume of 0.1 ml, or a weight of not more than 100 mg
(the weight must always be recorded). If the test material is solid or
granular, it should be ground to a fine dust. The volume of particulates
should be measured after gently canpacting thes, e.g., by tapping the
measuring container. To test a substance contained in a pressurized aerosol
container the eye should be held open and the test substance administered
in a single burst of about one second fran a distance of 10 an directly in
front of the eye. The dose may be estimated by weighing the container
before arid after use. Care should be taken not to damage the eye. Pump
sprays should not be used but instead the liquid should be expelled and
0.1 ml collected and instilled into the eye as described for liquids.
(3) Examination of eyes prior to test . (1) Both eyes of eath experi-
mental animal provisionally selected for testing should be examined within
24 hours before testing starts by the same procedure to be used during the
test examination. Animals showing eye irritation, ocular defects or pre-
existing cornea]. injury should not be used.
(4) Application of the test substance . (i) The test substance
should be placed in the conjunctival sac of one eye of eath animal after
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gently pulling the lower lid away from the eyeball. The lids are then
gently held together for about one second in order to prevent loss of the
material. The other eye, wh idi remains un trea ted, serves as a control.
If it is thought that the substance could cause extreme pain, a local
anesthetic may be used prior to instillation of the test substance. The
type and concentration of the local anesthetic should be carefully selected
to ensure that no significant differences in reaction to the test substance
will result fran its use. The control eye should be similarly anesthetized.
(ii) The eyes of the test animals should not be washed out for 24
hours following instillation of the test substance. At 24 hours, a washout
may be used if considered appropriate.
(5) Observation period . Ci) The duration of the observation period
should not be fixed rigidly, but it should be sufficient to evaluate fully
the reversibility or irreversibility of the effects observed. It normally
need not exceed 21 days after instillation.
(6) Clinical examination and scoring . (i) The eyes should be examined
at 1, 24, 48, and 72 hours. If there is no evidence of irritation at 72
hours, the study may be ended. Extended observation may be necessary if
there is persistent corneal involvement or other ocular irritation in
order to determine the progress of the lesions and their reversibility or
irreversibility. In addition to the observations of the cornea, iris
and conjunctivae, any other lesions thich are noted should be recorded and
reported. The grades of ocular reaction using Table 1 should be recorded
at each examination.
TABLE 1: GRADES FOR (XUIAR LESIONS
CORNEA
Opacity : degree of density (area most dense taken for reading).
— No ulceration or opacity . . . . . . 0
— Scattered or diffuse areas of opacity (other than slight dulling
of normal luster), details of iris clearly visible . 1*
— Easily discernible translucent area, details of iris slightly
o .. .scur ... . . . . . . . . . . . .
— Nacrous area, no details or iris visible, size of pupil barely
discernible . . . . . . . . . . . . . . . . . • . 3*
— Opaque cornea, iris nob discernible through the opacity 4*
*St red figures indicate positive effect.
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IRIS
— Normal . . 0
— Markedly deepened rugae, congestion, swelling, moderate circum—
corneal hyperemia, or injection, any of these or combination
of any thereof, iris still reacting to light (sluggish reaction
is positive) . . . . . . . . . . . . . . . . . . . . . . . . . . 1*
- No reaction to light, hemorrhage, gross destruction (any or
all of these) . . . . . . . . . . . . . . . . . . . . . . . . . . 2*
CONJUNCTIVAE
Redness (refers to palpebral and bulbar conjunctivae, cornea, and iris)
— Bloodvesselsnormal . 0
- Some blood vessels definitely hyperemic (injected) . . . . . . . . 1
— Diffuse, crimson color, individual vessels not easily discernible • 2*
— Diffuse beefy red • • • • • • • • • • . . . . . . . . . . • • • • 3*
Chemosis : lids and/or nictating membranes
— No swelling . . . . . . . . . . . . . . . . . • • • • • • • • . . . 0
- Any swelling above normal (includes nictating membranes) . . . . . 1
- Obvious swelling with partial eversion of lids 2*
— Swelling with lids about half closed . . . . . . . . . . . • • • • 3*
— Swelling with lids more than half closed 4*
Discharge
— No discharge . . . . . . . . • . . 0
— Any amount different from normal (does not include small amounts ob-
served in inner canthus of normal animals) . . . . . . • . . . . . . 1
— Discharge with moistening of the lids and hairs just adjacent to lids 2
- Discharge with moistening of the lids and hairs, and considerable
area around the eye 3
(ii) Examination of reactions can be facilitated by use of a binocular
loupe, hand slit—lamp, biomicroscope, or other suitable device. After record-
ing the observations at 24 hours, the eyes of any or all rabbits may be fur-
ther examined with the aid of fluorescein.
(iii) The grading of ocular responses is subject to various interpreta-
tions. To promote harmonization and to assist testing laboratories and those
involved in making and interpreting the observations, an illustrated guide in
grading eye irritation should be used. (Such an illustrated guide is in use
in the United States and can be obtained from the Consumer Product Safety
Commission, Washington, D.C. 20207.)
*Starred figures indicate positive effect.
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(g) Data and reporting . (1) Data shall be summarized in tabular form,
showing, for each individual animal:
(i) The irritation scores at the designated observation time;
(ii) A description of the degree and nature of irritation;
(iii) The presence of serious lesions; and
(iv) Any effects other than ocular which were observed.
(2) Evaluation of the results . The ocular irritation scores shall be
evaluated in conjunction with the nature and reversibilty or otherwise of
the responses observed. The individual scores do not rqresent an absolute
standard for the irritant properties of a material. They shall be viewed
as reference values and are only meaningful when supported by a full descrip-
tion and evaluation of the observations.
(3) Test report . In addition to the information required by § 80-4,
the test report shall include the following information:
(i) Physical nature and, where appropriate, concentration and pH value
for the test substance;
(ii) Species and strain used;
(iii) Tabulation of irritant/corrosive response data for each indivi-
dual animal at each observation time point (e.g., 1, 24, 48,
and 72 hours);
(iv) Description of any lesions observed;
(v) Narrative description of the degree and nature of irritation or
corros ion observed;
(vi) Description of the method used to score the irritation at 1, 24,
48, and 72 hours (e.g., hand slit—lamp, bicinicroscope, fluorescein); and
(vii) Description of any non—ocular effects noted.
§ 81—5 Primary dermal irritation .
(a) When required . Data on primary dermal irritation are required by
40 CFR Part 158 to support the registration of each manufacturing—use product
and each end—use product. See, specifically, 40 CFR § 158.50 and § 158.135
to determine whether these data must be subsiitted. Section Il—A of this
subdivision contains an ad di tio nal discussion of the “Formulators’
Exesiption” and who must suh it the required data as a general rule.
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(b) Purpose . In the assessment and evaluation of the toxic tharacter-
istics of a substance, determination of the irritant and/or corrosive effects
on skin of mammals is an important initial step. Information derived fran
this test serves to indicate the existence of possible hazards likely to
arise fr cxn exposure of the skin to the tes t subs tance.
(C) Definitions . (1) “Dermal corrosion” is the production of irrever-
sible tissue damage in the skin following the application of the test
substance.
(2) “Dennal irritation” is the production of reversible inflammatory
thanges in the skin following the application of a test substance.
(d) Principle of the test method . (1) The substance to be tested
is applied in a single dose to the skin of several experimental animals,
each animal serving as its own control. The degree of irritation is read
and scored at specified intervals and is further described to provide a
cosplete evaluation of the effects. The duration of the study should be
sufficient to permit a full evaluation of the reversibility or irreversibi-
lity of the effects observed but need not exceed 14 days.
(2) When testing solids (which may be pulverized if considered
necessary), the test substance should be moistened sufficiently with water
or, where necessary, a suitable vehicle, to ensure good contact with the
skin. When vehicles are used, the influence of the vehicle on irritation of
skin by the test substance should be taken into account. Liquid test
substances are generally used undiluted.
(3) Strongly acidic or alkaline substances, for example with a
demonstrated pH of 2 or less or 11.5 or greater, need not be tested for
primary derrnal irritation, owing to their predictable corrosive properties.
(4) The testing of materials which have been shown to be highly toxic
(LD5O less than 200 mqjkg) by the derma]. route is unnecessary.
(e) Substance to be tested . (1) Test substance . Ci) The manufac-
turing-use product shall be tested to support the registration of a manu-
facturing—use product.
(ii) The end-use product shal 1 be tes ted to support the registration
of an end—use product.
(2) Condition of test substance . Ci) If the substance is a liquid,
it should be applied undiluted.
(ii) If the test substance is a solid, it should be slightly moistened
with water or, where necessary, a suitable vehicle before application.
(3) Corrosive pesticides . Data which demonstrate that the test
substance specified by paragraph (e)(1) of this section has a pH of 1—2 or
11.5—14 may be submitted in lieu of data fran a primary dermal irritation
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study conducted in accordance with paragraph (f) of this section. For all
regulatory purposes, the Agency will assume that such a substance is
corrosive.
(f) Test procedures . (1) Animal selection . (i) Species and
strain . The albino rabbit is recanmended as the preferred species. If
another mammalian species is used, the tester should provide justification!
reasoning for its selection.
(ii) Number of animals . At least six healthy adult animals should be
used, unless justification/reasoning for using fewer animals is provided.
(2) Dose level . (i) A dose of 0.5 ml of liquid or 0.5 g of solid
or semi—solid is applied to the test site.
(ii) Separate animals are not recanmended for an untreated control
group. Adjacent areas of untreated skin of each animal serve as control
for the test.
(3) Preparation of animal skin . Approximately 24 hours before the
test, fur should be removed from the test area by clipping or shavi.ng
the dorsal area of the trunk of the animals. Care should be taken to avoid
abrading the skin. Only animals with healthy intact skin should be used.
(4) Application of the test substance . Ci) Exposure duration is for
four hours. Longer exposures may be indicated under certain conditions,
e.g., expected pattern of human use and exposure. At the end of the expo-
sure period, residual test substance should generally be removed, where
practicable, using water or an appropriate solvent, without altering the
existing response or the integrity of the epidermis.
(ii) The test substance should be applied to a small area (approxi-
mately 6 cm 2 ) of skin and covered With a gauze patch, which is held in
place with non—irritating tape. In the case of liquids or some pastes, it
may be necessary to apply the test substance to the gauze patch and then
apply that to the skin. The patch should be loosely held in contact with
the skin by means of a suitable semi-occlusive dressing for the duration
of the exposure period. However, the use of occlusive dressing may be
considered appropriate in some cases. Access by the animal to the patch
and resultant ingestion/inhalation of the test substance should be prevented.
(5) Observation period . (i) The duration of the observation period
is at least 72 hours, but should not be fixed rigidly. It should be suff i-
cient to evaluate fully the reversibility or irreversthlity of the effects
observed. It need not normally exceed 14 days after application.
(6) Clinical examination and scoring . (i) After removal of the
patch, animals should be examined for signs of erytheina and edema and the
responses scored within 30—60 minutes, and then at 24, 48 and 72 hours
after patch removal.
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(ii) Dermal irritation is scored and recorded according to the grades
in Table 2, below. Further observations may be needed, as neces-
sary, to establish reversibility. In addition to the observation
of irritation, any lesions and other toxic effects should be
fully described.
TABLE 2: EVALUATION OF SKtN REACt’ ION
Erythema and Eschar Formation Value
No erythema . . . . . . . . . . . . . . 0
Very slight erythema (barely perceptible) . . . . 1
Well—defined erythema . . . . . . . . . 2
Moderate to severe erythema . . . . . . 3
Severe erytheina (beet redness) to slight eschar
formation (injuries in depth) . . . . . . . 4
Maximum possible - 4
Edema Formation Value
No edema . . . . . . . . . . . . . . . . . . . . 0
Very slight: edema (barely perceptible) 1
Slight edema (edges of area well defined by
definite raising) . . . . . . . 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised r iore than 1 millimeter
and extending beyond area of exposure . . . 4
Maximum possible - 4
(g) Data and reporting . (1) Data shall be summarized in tabular
form, showing, for each individual animal:
Ci) The irritation scores for erythema and edema at 30 to 60 minutes,
24, 48, and 72 hours after patch renoval;
(ii) Any lesions;
(iii) A description of the degree and nature of irritation, corrosion,
or reversiblity; and
(iv) Any other toxic effects observed.
(2) Evaluation of results . The dermal irritation scores shall be
evaluated in conjunction with the nature and reversibility or otherwise of
the responses observed. The individual scores do not represent an absolute
standard for the irritant properties of a material. They should be viewed
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as reference values whith are only meaningful when supported by a full
description and evaluation of the observations • The use of an occlusive
dressing is a severe test and the results are relevant to very few likely
human cposure conditions.
(3) Test report . In addition to the information recanmended by § 80—4,
the test report should include the following information:
(i) Physical nature and, where appropriate, concentration and pH
value for the test substance;
(ii) Species and strain used;
(iii) Tabulation of irritation response data for each individual
animal for eath observation time period (e.g., 30 to 60 minutes, 24, 48, and
72 hours after patch removal);
(iv) Description of any lesions observed;
(v) Narrative description of the degree and nature’of irritation
observed; and
(vi) Description of any toxic effects other than dermal irritation.
81—6 Derinal sensitization study .
(a) When required . Data fran a dermal sensitization study are required
by 40 CFR Part 158 to support the registration of each manufacturing—use product
and of each end-use product which will result in repeated human skin contact
under conditions of use.
(1) See, specifically, 40 CFR § 158.50 and § 158.135 to de termine
whether these data must be subsitted. Section lI-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and who
must subsi t the required data as a general rule.
(b) Purpose . In the assessment and evaluation of the toxic tharacter-
istics of a substance, determination of its potential to provoke skin sensi-
tization reactions is important. Information derived fran tests for skin
sensitization serves to identify the possible hazard to a population
repeatedly exposed to the test substance. While the desirability of skin
sensitization testing is recognized, there are sane real differences of
opinion about the best method to use. The test selected should be a reliable
screening procedure whith should nob fail to identify substances with signi-
ficant allergenic potential, while at the seine time avoiding false negative
results.
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Cc) Definitions . (1) “challenge exposure” is an experimental exposure
of a previously treated subject to a test substance following an induction
period, to determine whether the subject will react in a hypersensitive
manner.
(2) “Induction expos ur e” is an expe imental ex pos ur e of a subj e Ct to
a test substance with the intention of inducing a hypersensitive state.
(3) “Induction period” is a period of at least one week following a
sensitization exposure during thid a hypersensitive state is developed.
(4) “Skin sensitization” (“allergic contact dermatitis”) is an
immunologically—mediated cutaneous reaction to a substance. In the human,
the responses may be tharacterized by pruritis, erythema, edema, papules,
vesicles, bullae, or a combination of these. In other species, the reactions
may differ and only erythema and edema may be seen.
(d) Principle of the test method . Following initial exposure(s) to a
test substance, the animals are subsequently subjected, after a period of
not less than one week, to a thallen exposure. with the test substance to
establish whether a hypersensitive state has been induced. Sensitization
is determined by examining the reaction to the thallenge exposure and
comparing this reaction to that of the initial induction exposure.
(e) Substance to be tested . (1) Test substance . (i) The manufac-
turing—use product shall be tested to support the registration of a
nianu acturing—use product.
(ii) The end—use product shall be tested to support the registration
of an end—use product.
(2) conditions of test substance . The test substance should be applied
at a concentration in accordance with the test methods. If the test substance
causes marked irritation, it should be diluted with physiological saline
until a concentration is found whith produces only sli t irritation. If
the test substance is a solid to be injected intradermally, it should be
dissolved in a minimum amount of physiological saline or suspended in a
suitable agent.
(f) Test procedures . (1) Any of the following seven test methods
is considered to be acceptable. It is realized, however, that the methods
differ in their probability and degree of reaction to sensitizing substances.
(i) Freurid’s canplete adjuvant test;
(ii) Guinea pig maximization test;
(iii) Split adjuvant technique;
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(iv) Buehler test;
(v) Open epicutaneous test;
(vi) Mauer optimization test; and
(vii) Footpad technique in guinea pig.
(2) Removal of hair is by clipping, shaving, or possibly by depilation,
depending on the test method used.
(3) Animal selection . (1) Species and strain . The young adult
guinea pig is the preferred species. Commonly—used laboratory strains
should be employed. If other species are used, the tester should provide
justification/reasoning for their selection.
(ii) Number and sex . (A) The number and sex of animals used should
depend on the method employed.
(B) The females should be nulliparous and non-pregnant.
(4) Control animals . (i) Periodic use of a positive control
substance with an acceptable level of reliability for the test system
selected is recommended.
(ii) Animals may act as their own controls or groups of thduced
animals can be compared to groups which have received only a challenge
exposure.
(5) Dose levels . (1) The dose level will depend upon the method
selected.
(6) Observation of animals . (i) Skin reactions are to be graded
and recorded after the challenge exposures at the time specified by the
methodology selected. This is usually 24, 48, and 72 hours. Additional
notations should be made as necessary to fully describe unusual responses.
(ii) Regardless of method selected, initial and terminal body weights
are to recorded.
(7) Procedures . (1) The procedures to be used are those described
by the methodology chosen.
(g) Data and reporting . (1) Data summary . Data should be summarized
in tabular form, showing, for each individual animal:
(i) The skin reaction; and
(ii) Results of the induction exposure(s) and the challenge exposure(s)
at the times indicated by the method chosen.
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(2) Grading information . As a minimum, the erythema and/or edema should
be graded and any unusual findings should be recorded.
(3) Evaluation of the results . The evaluation of results will provide
information on the proportion of each group that became sensitized and the
extent (slight, moderate, severe) of the sensitization reaction in each
individual animal.
(4) Test report . In addition to the information required by § 80-4,
the test reports shall include the following information:
(i) A description of the method used and the canmonly accepted name;
(ii) Information on positive control study, including:
(A) Positive control used;
(B) Method used; and
(C) Time conducted.
(iii) The number, species, strain and sex of the test animals;
(iv) Individual weights of the animals at the start of the test and at
the conclusion of the test;
(v) A brief description of the grading system; and
(vi) Eath reading made on eath individual animal.
81—7 Acute Delayed Neurotoxicity of Organophosphorus Substances .
(a) Introduction, Purpose, Scope, Relevance, Application and Limits _
of Test . (1) When Required . As stated in 40 CFR Part 158, organophosphorous
substances should be considered as candidates for delayed neurotoxicity
studies using the adult hen as the test animal. This test has certain
limitations, e.g., in predicting effects from repeated exposures. These
limitations may possibly be minimized by conducting an adjunct test in
whidi the inhibition and aging of neurotoxic esterase of hen neural tissue
are measured.
(i) See, specifically, 40 CFR 158.50 and § 158.135 to determine
whether these data must be subsitbed. Section Il—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must suhnit the required data as a general rule.
(2) Purpose . This screening procedure is for detecting delayed
neuro toxic potential.
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(3) Definition . “Acute delayed neurotoxicity” is a prolonged,
delayed—onset locanotor ataxia resulting fran single administration of the
test substance, repeated once if necessary.
(4) Substance Tested . The tethnical grade of the active ingredient.
(5) Reference Substances . A substance ith is known to produce
acute delayed neurotoxicity should be used as a positive control. Examples
of suth substances are tri—orthocresyl phosphate (TOCP) and leptophos.
(6) Principle of the Test Method . The test substance is administered
orally in a single dose to danestic hens ( Gallus gallus doinesticus ) thich
have been protected from acute tholinergic effects. The animals are ob-
served for at least 21 days for delayed neurotoxicity, with redosing and
observation for another 21 days if no effects or eguivocal responses are
seen. The animals are observed daily for behavioral abnormalities, locomo-
tor ataxia and paralysis. Histopathological examination of selected neural
tissues is undertaken on all animals surviving the initial cholinergic
phases.
(b) Description of the Test Procedure . (1) Preparations .
A preliminary LD5O test using an apprvpriate number of animals, dosages
and dose groups, should be performed in unprotected hens to establish the
dose level to be used in this test. Healthy young adult hens free fran
interfering viral diseases and medication and without abnormalities of
gait should be acclimatized to the laboratory conditions for at least five
days prior to randomization and assignment to treatment and control groups.
(2) Experimental Animals . (i) Selection of Species . The adult
domestic laying hen, aged between 8-14 months, is recommended. Standard
size breeds and strains should be employed. -
(ii) Number . A sufficient number of hens should be utilized so
that at least six survive the observation period.
(iii) Controls . Appropriate control groups should be used. These
should include a positive control group of a least four hens treated with
a known delayed neurotoxicant and a concurrent negative control group of
at least six hens treated in a manner identical to the test group, except
that administration of the test substance and any protective agents is
omit ted.
(iv) Housing and Feeding Conditions . Cages or enclosures c ich are
large enough to permit free mobility of the hens and easy observation of
gait should be used. Where the lighting is artificial, the sequence
should be 12 hours light, 12 hours dark. Appropriate diets should be
administered as well as an unlimited supply of drinking water.
(3) Test Conditions . Ci) Dose Levels . The selected dose level
of the test substance should not be less than the unprotected LD5O dose.
Atropine or another protective agent demonstrated to be non—interfering
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64
may be used to prevent death due to acute cholinergic effects. Doses of
test substance higher than 5000 mg/kg of body weight need not be tested.
(ii) Route of Administration . Dosing with the test substance should
normally be by the oral route using gavage or gelatine capsules.
(4) procedure . The test or control substance should be administered
and observations begun. All hens should be carefully observed at least
once daily for a period of at least 21 days and signs of toxicity recorded,
including the time of onset, degree and duration. Observations should
include, but not be limited to, behavioral abnormality, locomotor ataxia
and paralysis. At least twice a week the hens should be taken outside the
cages and subjected to a period oi forced motor activity, such as ladder
climbing, in order to enhance the observation of minimal responses. If
neurotoxic responses are not observed or if equivocal responses are seen,
then the dose should be administered again and the animals observed for an
additional 21 days. The hens should be weighed weekly. Any moribund
hens should be removed and sacrificed.
(5) Pathology . Ci) Gross Necropsy . Useful information is not
usually provided by the results of gross necropsy.
(ii) Histopathology . All animals should be subjected to microscopic
examination. Tissues should be fixed in situ , preferably using perfusion
techniques. Sections should include medulla oblongata, spinal cord and
peripheral nerves. The spinal cord sections should be taken from the
upper cervical bulb, the midthoracic and the luxnbo—sacral regions. Sections
of the proximal region of the tibial nerve and its branches should be
taken. Sections should be stained with appropriate myelin and axon—specific
stains.
(C) Data and Reporting . (1) Treatment of Results . Data shall be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, the number of animals showing lesions or
effects, the types of lesions or effects and the percentage of animals
displaying each type of lesion or ffect.
(2) Evaluation of Results . The findings of an acute delayed neuro-
toxicity study shall be evaluated in terms of the incidence and severity
of neurotoxic effects and of any other observed effects and histopathological
findings in the treated and control groups.
(3) Test Report . The test report shall include the following
information:
(1) Toxic response data by group with a description of clinical
manifestations of nervous system damage; where a grading system is used
the criteria should be defined.
(ii) For each animal, time of death during the study or whether
it survived to termination.
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(iii) The day of observation of eath abnormal sign and its sub-
sequent: course.
(iv) Body weight: data.
(v) Necropsy findings for each animal, hen performed.
(vi) A detailed description of all histopathological findings.
(vii) Statistical treatment of results, ere appropriate.
(4) Interpretation of Results . This study provides information on
the acute delayed n ir otoxic effects of exposure to orga nopho s pho ru s sub-
stances. Extrapolation from the results of the study to man is valid only
to a limited degree, although it can prov-ide useful information on the
degree of neurotoxic activity of a substance.
(5) Literature .
(1) M.B. AbQ.i—Donia, Ann. Rev. Pharmacol. Toxicol 21 , 511—548 (1981).
(2) M.B. Abou—Donia and S.H. Pressing, Toxicol. Appi. Pharmacol. 38 ,
5995—6008 (1976).
(3) EPA—600/176—025 (edited by R.L. Baron), (National Teth. Info.
Services, Springfield, VA., 1976).
(4) British Working Documents, October, 2 (Ministry of Agriculture,
Fisheries and Food, London, 1967).
(5) J.B. Cavanaugh, CRC Critical Review of Toxicity 2 (3),
365—417 (1973).
(6) F.R. Johannsen, P.L. Wright, D.E. Gordon, G.L. Levinskas,
R.W. Radue and P.R. Graham, Toxicol. Appl. Pharmacol. 41 ,
291—304 (1977).
(7) M.K. Johnson, Arch. Toxicol. 34 , 259—288 (1975).
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Series 82: SUBCHPONIC TESTING
(NOTE: The sections of this series are prepared in conformity with the
guidelines developed by the Organization of Economic Cooperation and
Development. Those guidelines were adapted t fit the toxicology data
requirements under FIFRA.]
§ 82-1 Subchronic oral toxicity (rodent and non—rodent): 90—day study .
(a) When required . As required by 40 CFR Part 158 data from subchronic
oral dosing studies are needed to support the registration of each
manufacturing—use product and end-use product that meet either of the
following criteria:
(1) The use for which registration application is made requires a
tolerance for the pesticide or an exes ption from the requirement to obtain
a tolerance, or requires the issuance of a food additive regulation; or
(2) The use of the pesticide product is likely to result in repeated
human cposure to the product, its active ingredient(s), metabolite(s), or
degradation product(s) through the oral route.
(3) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be su)mtitted. Section lI-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and who
must su xnit the required data as a general rule.
(b) Purpose . En the assessment and evaluation of the toxic d aracter-
istics of a themical, the determination of subchronic oral toxicity may be
carried out after initial information on toxicity has been obtained by
acute testing. The subchronic oral study has been desigeed to permit the
determination of the no—observed—effect level and toxic effects associated
with continuous or repeated exposure to a substance for a period of 90
days. The test is not capable of determining those effects that have a long
latency period for development (e.g., carcinogenicity and life shortening).
It provides information on possible health hazards likely to arise frati
repeated exposures over a limited period of time. It should provide informa-
tion on target organs, the possibilities of accumulation, and can be of
use in selecting dose levels for chronic studies and for establishing
safety criteria for human exposure.
Cc) Definitions . (1) “Cumulative toxicity” is the adverse effects
of repeated doses occuring as a result of prolonged action on, or increased
concentration of the administered substance or its metabolites in, suscep-
tible tissue.
(2) “Dose” is the amount of test substance administered. Dose is
expressed as weight of test substance (g, mg) per unit weight of test animal
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(e.g., ing/kg), or as weight of test substance per unit weight of food or
drinking water.
(3) “No—effect level”/”No—toxic—effect level”/”No—adverse—effect
level”/”No—observed—effect level” is the maximum dose used in a test which
produces no observed adverse effects. A no—observed-effect level is
expressed in terms of the weight of a substance given daily per uni t weigh t
of test animal (mg/kg). When administered to animals in food or drinking
water the no-observed-effect level is expressed as mg/kg of food or mg/ml
of water.
(4) “Subthronic oral toxicity” is the adverse effects occurring as
aresult of the repeated daily oral dosing of a themical to experimental
animals for part (approximately ten percent) of a life span.
(d) Principle of the test method . The test substance is administered
orally in graduated daily doses to several groups of experimental animals,
one dose level per group, for a period of at least 90 days. During the
period of administration, the animals are observed daily to detect signs
of toxicity. Animals which die during the period of administration are
rtecropsied, and at the conclusion of the test, all surviving animals are
sacrificed and necropsied, and appropriate histopathological examination
is carried out.
(e) Substance to be tested . Testing shall be performed with the
technical grade of eadi active ingredient in the product.
(f) Limit test . If a test at one dose level of at least 1000 mg/kg
body weight (expected human exposure may indicate the need for a high dose
level), using the procedures described for this study, produces no observ-
able toxic effects and if toxicity would not be expected based upon data
of structurally—related compounds, then a full study using three dose
levels might not be necessary.
(g) Test procedures . (1) Animal selection . Ci) Species and strain .
A variety of rodent species may be used, although the rat is the preferred
species. Commonly used laboratory strains should be employed. The commonly
used non—rodent species is the dog, preferably of a defined breed; the
beagle is frequently used. If other mammalian species are used, the tester
should provide justification/reasoning for their selection.
(ii) . (A) General . Young, healthy animals should be employed.
At the commencement of the study, the weight variation of animals used
should not exceed t 20 percent of the mean weight.
(B) Rodents . Dosing should begin as soon as possible after weaning,
ideally before the rats are 6 and, in any case, not more than 8 weeks old.
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(C) Non—rodents . In the case of the dog, dosing should be cat menced
after acclimatization, preferably at 4—6 n nths and not later than 9 n nths
of age.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) The femal. es should be nul lipa rous and non-pregnant.
(iv) Numbers . (A) Rodents . At least 20 animals (10 females and 10
males) should be used at each dose level.
(B) Non—rodents . Eight animals (4 female and 4 male) should be used
at each dose level.
(C) Allowance for sacrifice . If interim sacrifices are planned, the
number should be increased by the number of animals seheduled to be sacri-
ficed before canpietion of the study.
(D) Number at termination of study . The number of animals at the
termination of the study must be adequate for a meaningful evaluation of
toxic effects.
(2) Control groups . A concurrent control group is recanmended. This
group should be an untreated or sham treated control group or, if a vehicle
is used in administering the test substance, a vehicle control group. If
the toxic properties of the vehicle are not known or cannot be made avail-
able, both untreated and vehicle control groups are required.
(3) Satellite group (rodent) . A satellite group of 20 animals (10
animals per sex) may be treated with the high dose level for 90 days and
observed for reversibility, persistence, or delayed occurrence of toxic
effects for a post—treatment period of appropriate length, normally not
less than 28 days.
(4) Dose levels and dose selection . Ci) In subcthronic toxicity
tests, it is desirable to have a dose response relationship as well as
a no observed toxic effect level. Therefore, at least three dose levels
and a control should be used. Where appropriate, a vehicle control (cor-
responding to the concentration of vehicle at the highest exposure level)
should be used.
(ii) The highest dose level in rodents should result in toxic
effects but not produce an incidence of fatalities sthich would prevent
a meaningful evaluation; for nonridents, there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of toxi-
city. Where there is a usable es tuna tb n of human exposure, the lowes t
level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable effects. If n re than one intermediate dose is used, the dose
levels should be spaced to produce a gradation of toxic effects.
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(v) For rodents, the incidence of fatalities in the low and inter-
mediate groups and in the controls should be low, to permit a meaningful
evaluation of the results. For non—rodents, there should be no fatalities.
(5) Exposure conditions . The animals are treated with test substance
ideally for on a 7—days—per—week basis, for a period of 90 days. However,
based primarily on practical considerations, dosing in gavage or capsule
studies on a 5—days—per—week basis is considered to be acceptable.
(6) Observation period . Ci) Duration of observation should be for
at least 90 days.
(ii) Animals in the satellite group, if included, scheduled for
follow—up observations should be kept for a further 28 days without treat-
ment to detect recovery fran, or persistence of, toxic effects.
(7) Administration of the test substance . Ci) The test substance
may be administered in the diet or in capsules. In addition, for rodents,
it may also be administered by gavage or in the drinking water.
(ii) All animals should be dosed by the same method during the entire
experimental period.
(iii) Where necessary, the test substance is dissolved or suspended
in a suitable vehicle. If a vehicle or diluent is needed, ideally it
should not elicit important toxic effects itself nor substantially alter
the chemical or toxicological properties of the test substance. It is
recanmended that, whenever possible, the use of an aqueous solution be
considered first, followed by consideration of a solution of oil, and then
by possible solution in other vehicles. For non-aqueous vehicles, the toxic
characteristics should be known, and if not known, should be determined
before the test.
(iv) For substances of low toxicity, it is important to ensure that,
when administered in the diet, the quantities of the test substance involved
do not interfere with normal nutrition. When the test substance is admin-
istered in the diet, either a constant dietary concentration (ppm) or a
constant dose level in terms of the animal’s body weight, may be used;
the alternative used should be specified.
(v) For a substance administered by gavage or capsule, the dose should
be given at similar times each day, and adjusted at intervals (weekly or
biweekly) to maintain a constant dose level in terms of animal body weight.
(8) Observatio n of animals .
(1) A careful cageside camination should be made at least once each
day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
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refrigeration of those animals found dead, and isolation or sacrifice of
weak or moribund animals, to ensure that not more than 10% of the animals
in any study group are lost from the test due to cannibalism, analysis of
tissues, misplacement, and similar management problems.
‘iii) Clinical signs of toxicity should be recorded as they are
observed including the time of onset, degree and duration.
(iv) Cageside observations should include, but not be limited to,
changes in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Autonemic and central nervous system;
(F) Somatoniotor activity; and
(G) Behavior pattern.
(v) Measurements should be made weekly of food and water consumption,
depending on the mode of administration of the test substance.
(vi) Animals should be weighed weekly.
(vii) At the end of the 90-day period, all survivors in the non—
satellite treatment groups are sacrificed. Any moribund animals should be
removed and sacrificed when noticed.
(9) Clinical examinations .
Ci) The following examinations should be made on all animals of each
sex in each group:
(A) Hematology determinations which are considered to be appropriate
to all studies are:
(! ) Hematocrit;
(2) Hemoglobin concentration;
(3) Erythrocyte count;
(4) Total and differential leucocyte count; and
(5) A measure of clotting potential suth as clotting time, prothrombin
time, thranboplastin time, or platelet count.
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(B) Hematology determinations should be investigated for non—rodents
at the beginning, then either at n nthly intervals or midway through the
test period, and, finally, at the end of the test period; for rodents,
determinations should be investigated at the end of the test period only.
(C) Clinical biothemistry determinations on blood should be carried
out for rodents at the end of the test period, and for non-rodents at
the beginning, then either at monthly intervals or midway through the test
period, and finally at the end of the test period. Test areas whith are
considered appropriate to all studies are electrolyte balance, carbthydrate
metabolism, liver and kidney function. The selection of specific tests
will be influenced by observations on the mode of action of the substance.
Non-rodents should be fasted for a period (not n re than 24 hours) before
taking blood samples. Suggested determinations are:
(1) Calcium;
(a.) Phosphorus;
(3) Chloride;
( ) Sodium;
(5) Potassium;
(6) Fasting glucc6e (with period of fasting appropriate to the species);
(7) Serum glutamic—pyruvic transaminase (also known as serum alanine
aminotrans ferase);
(8) Serum glutamic-oxaloacetic transaxninase (also known as serum
aspartite aminotransferase);
(!) Urea nitrogen;
(10) Albumen;
(11) Blood creatinine;
(12) Total bilirubin; and
(13) Total serum protein measurements.
(14) Other determinations thich may be necessary for an adquate toxi-
cological evaluation include analyses of lipids, hormones, acid/base balance,
methemoglobin, and cholinesterase activity.
(15) Additional clinical biotheniistry may be employed, there necessary,
to extend the investigation of observed effects.
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(ii) The following examinations should be made on high dose and control
animals of eath sex in each group:
(A) Opthalmological examination, using an opthalmoscope or equivalent
suitable equipment. This examination should be made prior to the adminis-
tration of the test substance and at the termination of the study,
preferably in all animals, but at least in the high dose and control groups.
If changes in the eyes are detected all animals should be examined.
(B) Urinalysis is not required on a routine basis but only when there
is an indication based on expected or observed toxicity.
(10) Gross necropsy .
(i) All animals should be subjected to a full gross necropsy which
includes examination of:
(A) The external surface of the body.
(ii) At least the liver, kidneys, and testes should be weighed wet
as soon as possible after dissection to avoid drying. In addition, for
the non-rodent, the thyroid with parathyroids should be weighed wet.
(11) Tissue preservation .
(i) The following organs and tisst s, or representative samples
thereof, should be preserved in a suitable medium for possible future
his topa tho logical examination:
(A) All gross lesions;
(B) Brain — including sections of medulla/pons, cerebellar cortex, and
cerebral cortex;
(C) Pituitary;
(D) Thyroid parathyroid;
(E) Thymus;
(F) Lungs, trachea;
(G) Heart;
(H) Bone marrow (either femur, sternum or rib at the costodiondral
junction);
(I) Salivary glands;
(J) Liver;
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(K) Spleen;
CL) Kidneys;
CM) Adrenals;
(N) Pancreas;
(0) Testes;
(P) Uterus;
(Q) Aorta;
CR) Esophagus;
CS) StQnach;
(T) Duodenum;
(U) Jejunum;
(V) Ileum;
(W) Caecum;
(x) Colon;
(Y) Rectum;
(Z) Urinary bladder;
(AA) Representative lymph node;
(BB) Peripheral nerve; and
(CC) Gall bladder (if present).
(ii) The following tissues need be preserved only if indicated by signs
of toxicity or target organ involvement:
(A) Trachea;
(B) Sternum with bone marrow;
(C) Mammary gland;
(D) Thigh musculature;
(E) Eyes;
(F) Femur — including articular surface;
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(G) Spinal cord at three levels — cervical, midthoracic, and lumbar;
(H) Exorbital lachryrnal glands (rodent); and
(J) Gall bladder (non—rodent).
(12) Histopathology .
(i) The following histopathology should be performed:
(A) Full histopathology on the organs and tissues, llsted in paragraph
(g)(11) of this section, of all rodents in the control and high dose groups,
all non—rodents, and all rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) The following histopathology should be performed. Further histo-
pathological examination may not be recanmended on the animals in these groups,
but must always be carried out in organs ich showed evidence of lesions in
the high dose group. These organs should be preserved for future
histopathological examination.
(A) Lungs of rodents in the low and intermediate dose groups (special
attention to examination of the lungs of rodents should be made for evidence
of infection since this provides an assessment of the state of health of
the animals);
(B) Liver of all animals in the low and intermediate dose groups; and
(C) Kidneys of all animals in the low and intermediate dose groups.
(iii) For rodents, .hen a satellite group is used, histopathology
should be performed on tissues and organs identified as showing effects in
the treated groups.
(h) Data and reporting .
(1) Treatment of results .
(i) Data shall be summarized in tabular form, showing for each test
group the number of animals at the start of the test, the number of animals
showing lesions, the types of lesions and the percentage of animals display-
ing eadi type of lesion.
(ii) All observed results, quantitative and incidental, shall be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical method shall be selected
during the design of the study.
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(2) Evaluation of the study results .
(A) The findings of a subthronic oral toxicity study should be eval-
uated in conjunction with the findings of preceding studies and considered
in terms of the toxic effects and the necropsy and histopathological findings.
The evaluation should include the relationship between the dose of the test
substance and the presence or absence, the incidence and severity, of abnor-
malities, including:
(1) Behavioral abnormalities;
(2) Clinical abnormalities;
(3) Gross lesions;
(4) Identified target organs;
(5) Body weight thanges;
(6) Effects on mortality; and
(7) Any other general or specific toxic effects.
(8) A properly conducted subchronic test shall provide a satisfactory
estimation of a no—effect level.
(B) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considernd.
(3) Test report . In addition to the information r& uired by § 80—4,
the test report summary shall include the following information:
(i) Toxic response and other effects data by sex and dose;
(ii) Species, strain, and/or breed;
(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination;
(B)
(C)
(D)
(E)
(F)
Time of observation of each abnormal sign and its subsequent course;
Food or water consumption data;
Body weight data;
Results of ophthalmological examination;
Hematological tests and all results;
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(G) Clinical biochemistry tests and all results;
(H) Necropsy findings;
(I) Detailed description and classification of all histopathological
findings; and
(iv) Statistical method applied and treatment of results.
§ 82—2 Repeated dose dermal toxicity: 21 day study .
(a) When required . Data fran a subthronic 21-day dermal toxicity
study are required by 40 CFR Part 158 to support the registration of each
manufacturing-use product and each end—use product whose pesticidal use is
likely to result in repeated human skin contact with the product, its
active ingredients, or their breakdown products. Data fran this study are
not required when data from a subchronic 90—day derinal toxicity study (see
§ 82—3) are required.
(1) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be suhuitted. Section Il-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who mus t s uhni t the r equir ad da ta as a gener al rule.
(b) Purpose . In the assessment and evaluation of the toxic character-
istics of a chemical, the determination of subchronic dermal toxicity may
be carried out after initial information on toxicity has been obtained by
acute testing. It provides information on possible health hazards likely
to arise fr an repeated exposures by the derinal route over a limited period
of time.
Cc) Definitions .
(1) “Cumulative toxicity” is the adverse effects of repeated doses
occurring as a result of prolonged action on, or increased concentration
of the administered substance or its metabolities in susceptible tissues.
(1) “Dose” in a dermal test is the amount of test substance applied
to the skin. Dose is expressed as weight of test substance (g, mg) per
unit weight of test animal (e.g., mg/kg).
(2) “No—effect level”/”No—toxic—effect level”/”No—adverse—effect
level”/”No—observed—effect level” is the maximum dose used in a test which
produces no adverse effects. A no—effect level is expressed in terms of
the weight of a substance given daily per unit weight of test animal (mg/kg).
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(d) Principle of the test method . The test substance is applied
daily to the skin in graduated doses to several groups of experimental
animals, one dose per group, for a period of 21 days. During the period of
application the animals are observed daily to detect signs of toxicity.
animals which die during the test are necropsied, and at the conclusion of
the test the surviving animals are sacrificed and necropsied and appropriate
histopethological. examinations carried out.
(e) Substance to be tested . The ted nical grade of each active
ingredient in the product shall be tested. The end—use product shall also
be tested when any c nponent of the end—use product is likely to increase
dermal absorption of the test substance or potentiate toxic and pharmacologic
effects.
(f) Limit test . If a test at one dose level of at least 1000 mg/kg
body weight (but expected human exposure may indicate the need for a higher
dose level), using the procedures described for this study, produces no
observable toxic effects, and if toxici ty would not be expected based upon
data of structurally-related canpounds, then a full study using three dose
levels might not be necessary.
(g) Test procedures .
(1) Animal selection .
(i) Species and strain . The adult rat, rabbit or guinea pig may be
used. Other species may be used but their use would require justification.
(ii) . Adult animals should be used. The following weight ranges
at the start of the test are suggested in order to provide animals of a
size which facilitates the conduct of the test:
(A) Rats, 200 to 300 g.
(B) Rabbits, 2.0 to 3.0 kg.
(C) Guinea pigs, 350 to 450 g.
(iii) Sex. (A) Equal numbers of animals of each sex with healthy
skin should be used at each dose level.
(B) The females should be nulliparous and non-pregnant.
(iv) Numbers . At least 10 animals (5 females and 5 males) should be
used at each dose level.
(2) Control groups . A concurrent control group is recanmended. This
group should be an untreated or sham treated control group, or, if a vehicle
is used in administering the test substance, a vehicle control group. If
the toxic properties of the vehicle are not known or cannot be made avail-
able, both untreated and vehicle control groups are recanmended.
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(3) Satellite group . A satellite group of 10 animals (5 animals per
sex) may be treated with the high dose level for 21 days and observed for
reversibility, persistence, or delayed occurrence of toxic effects for 14
days post—treatment.
(4) Dose levels and dose selection . (i) At least three dose
levels, with a control and, where appropriate, a vehicle control, should
be used.
(ii) The highest dose level should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful
evalua tion.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the lowest
level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If n re than one intermediate dose is used the
dose levels should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups and in the controls the
incidence of fatalities should be low, to permit a meaningful evaluation o
the results.
(vi) If application of the test substance produces severe skin
irritation, the concentration may be reduced although this may result in a
reduction in, or absence of, other toxic effects at the high dose level.
However, if the skin has been badly damaged early in the study it may be
necessary to terminate the study and undertake a new study at lower
concen tra tions.
(5) Exposure conditions . The animals are treated with the test sub-
stance, ideally for at least 6 hours per day on a 7-days-per-week basis,
for a period of 21 days. However, based primarily on practical cons idera-
tions, application on a 5—days—per—week basis is considered to be acceptable.
(6) Observation period . Duration of observation should be for at
least 21 days.
(7) Preparation of animal skin . (i) Shortly before testing, fur
is clipped from the dorsal area of the trunk of the test animals. Shaving
may be employed but it should be carried out approximately 24 hours before
the test. Repeat clipping or shaving is usually needed at approximately
weekly intervals. When clipping or shaving the fur, care must be taken to
avoid abrading the skin which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be
clear for the application of the test substance. The weight of the animal
should be taken into account when deciding on the area to be cleared and on
the dimensions of the covering.
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(iii) When testing solids, c ich may be pulverized if appropriate,
the test substance should be moistened sufficiently with water or, s’there
necessary, a suitable vehicle to ensure good contact with the skin. When a
vehicle is used, the influence of the vehicle on penetration of skin by the
test substance should be taken into account.
(8) Application of the test substance . (i) The test substance
should be applied uniformly over an area whith is approximately 10 percent
of the total body surface area. With highly toxic substances the surface
area covered may be less, but as mudi of the area should be covered with
as thin and uniform a film as possible.
(ii) During the exposure period the test substance is held in contact
with the skin with a porous gauze dressing and nonirritating tape. The
test site should be further covered in a suitable manner to retain the
gauze dressing and test substance and ensure that the animals cannot ingest
the best substance. Restrainers may be used to prevent the ingestion of
the test substance but canplete inuxbilizabion is not a recanrnended method.
(9) Observation of animals . (i) A careful cageside examination
should be made at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of
weak or nxribund animals, to ensure that not nore than 10% of the animals
in any test group are lost fran the test due to cannibalism, analysis of
tissues, misplacement, and similar management problems.
(iii) Signs of toxicity should be recorded as they are observed
including the tiine of onset, the degree and duration.
(iv) Cageside observations should include, but not be limited to,
d ianges in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Aubonanic and central nervous system;
(F) Somatomotor activity; and
(G) Behavior pattern.
(v) Animals should be weighed weekly. Food consumption data should
be collected weekly.
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(vi) At the end of the study period all survivors in the non—satellite
treatment groups are sacrificed. Moribund animals should be removed and
sacrificed when noticed.
10) Clinical examinations . The following examinations should be made
on all animals:
Ci) Hematology, including:
(A) Hematocrit;
(B) Hen globin concentration;
(C) Erythrocyte count;
(D) Total and differential leucocyte count; and
(E) A measure of clotting potential such as clotting time, prothrombin
time, thromboplastin time, or platelet count, at the end of the test period.
(ii) Clinical biochemistry determinations on blood should be carried
out at the end of the test period. Blood parameters of liver and kidney
function are appropriate. The selection of specific tests will be influ-
enced by observations on the mode of action of the substance. Suggested
determinations are:
(A) Calcium;
(B) Phosphorus;
- (C) Chloride;
CD) Sodium;
CE) Potassium;
(F) Fasting glucose (with period of fasting appropriate to the species);
(G) Serum glutamic—pyruvic transaminase (also known as serum alanine
aminotransferase);
(H) Serum glutamic—oxaloacetic transaminase (also known as serum
aspartite aminotransferase);
(I) Urea nitrogen;
(3) Albumen;
(K) Blood creatinine;
CL) Total bilirubiri; and
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(M) Total serum protein measurements.
(N) Other determinations which may be necessary for an adequate
toxicological evalution include analyses of lipids, horn ,ries, acid/base
balance, methemoglobin, cholinesterase activity.
(0) Additional clinical biothemistry may be employed, where necessary,
to extend the investigation of observed effects.
(iii) Urinalysis is not required on a routine basis, but only when
there is an indication of obtaining useful data based on expected or observed
toxicity.
(11) Gross necropsy .
(i) All animals should be subjected to full gross necropsy which
includes examination of:
(A) The external surface of the body;
(ii) The liVer, kidneys and testes should be weighed wet as soon as
possible after dissection to avoid drying.
(12) Tissue preservation . The following organs and tissues, or
r resentative samples thereof, should be preserved in a suitable medium
for poss able future his topathological examination:
(i) Normal and treated skin;
(ii) Liver;
(iii) Kidney; and
(iv) Target organs (i.e., those organs showing gross lesions or
cthanges in size, whith are suspected to be related to the treatment of
the test substance).
(13) Histopathology . Histological examination should be performed on
the preserved organs and tissues of the high dose group and the control
group. These examinations should be extended to animals of other dosage
groups, if considered necessary to investigate the thanges observed in the
high dose group. Animals in the satellite group if included should be
examined histologically with particular emphasis on those organs and
tissues identified as showing effects in the other treated groups.
(h) Data and reporting . (1) Treatment of results . (i) Data
shall be summarized in tabular form, showing for each test group:
(A) The number of animals at the start of the test;
(B) The number of animals showing lesions;
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(C) The type of lesions; and
(D) The percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, shall be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical method shall be selected
during the design of the study.
(2) Evaluation of results . The findings of a subthronic dermal
toxicity study shall be considered in terms of the observed toxic effects
and the necropsy and histopathological findings.
Ci) The evaluation should include the relationship between the dose of
the test substance and the presence or absence, the incidence and severity,
of abnormalities, including:
(A) Behavioral abnormalities;
(B) Clinical abnormalities;
(C) Gross lesions;
(D) Identified target organs;
(E) Body weight thanges;
(F) Effects on mortality; and
(G) Any other general or specific toxic effects.
(ii) A properly conducted 21-day study should provide information on
the effects of repeated dezmal application of a substance and can indicate
the need for further longer term studies.
(iii) It can also provide information on the selection of dose levels
for longer term studies.
(3) Test report . In addition the information r uired by § 80-4,
the test report summary shall include the following information:
Ci) Toxic response and other effects data by sex and dose.
(ii) Species and strain.
(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination;
(B) The time of the obe erva tic n of each abnormal s ign and i ts subs e—
quent course;
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(C) Body weight data;
(D) Hematological tests employed and results;
(E) Clinical biothemistry tests employed and results;
(F) Necropsy findings;
(G) Detailed description of all histopathological findings; and
(iv) Statistical treatment of results, where appropriate.
§ 82—3 Subchronic dermal toxicity: 90—day study .
(a) When required . •jData from a subchronic 90—day dermal toxicity
study are required by 40 CFR Part 158 to support the registration of each
manufacturing—use product and end—use product whose use will involve
purposeful application to the human skin or whose pesticidal use will
result in canparable human exposure to the product, its active ingredients,
or their breakdown products (e.g., swimming pool algaecides, pesticides
for impregnating clothing), and ich meets either of the following criteria:
(1) Data fran a su]Dthronic oral study (see § 82—1) are not required; or
(2) The active ingredient of the product is known or expected to be
metabolized differently by the derinal route of exposure than by the oral
route, and a metabolite of the active ingredient is the toxic moiety.
(3) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be sulmdtted. Section II—1 of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must sulinit the required data as a general rule.
(b) Purpose . In the assessment and evaluation of the toxic tharacter-
is tics of a diemical the determination of subchronic dermal toxicity may
be carried out after initial information on toxicity has been obtained by
acute testing. The subchronic dermal study has been designed to permit the
determination of the toxic effects associated with continuous or repeated
exposure to a test substance for a period of 90 days. The test is not
capable of determining those effects that have a long latency period for
development (e.g., carcinogenicity and life shortening).
(C) Definitions . (1) “Cumulative toxicity” is the adverse effects
of repeated doses occurring as a result of prolonged action on, or increased
concentration of the administered substance or its metabolites, in susceptible
tissues.
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(2) “Dose” in a dermal test is the amount of test substance applied
to the skin (applied daily in subthronic tests). Dose is expressed as
weight of the test substance (g, mg) per unit weight of test animal (e.g.,
mg/kg).
(3) “No—effect level”/”No—toxic—effect level”/”No—adverse—effect
level”/”No—observed—effect level” is the maximum dose used in a test which
produces no adverse effects. A no—effect level is expressed in terms of
the weight of a substance given daily per unit weight of test animal
(mg/kg).
(4) “Subthronic dermal toxicity” is the adverse effects occurring as
a result of the repeated daily dermal application of a themical to experi-
mental animals for part (approximately 10 percent) of a life span.
(d) Principle of the test method . The test substance is applied
daily to the skin in graduated doses to several groups of experimental
animals, one dose per group, for a period of 90 days. During the period of
application the animals are observed daily to detect signs of toxicity.
Animals which die during the test are necropsied, and at the conclusion of
the test the surviving animals are sacrificed and necropsied, and appropriate
histopathological examinations carried out.
Ce) Substance to be tested . The ted nical grade of each active ingre-
dient in the product shall be tested. In addition, the end—use product shall
be tested if any cuitponent of the end-use product is likely to increase dermal
absorption of the test substance and potentiate toxic or pharmacologic effects.
(f) Limit test . If a test at one dose level of at least 1000 mg/kg
body weight (expected human exposure may indicate the need for a high dose
level), using the procedures described for this study, produces no observable
toxic effects and if toxicity would not be expected based upen data of
structurally—related canpounds, then a full study using three dose levels
might not be necessary.
(g) Test procedures . (1) Animal selection . (i) Species and
strain . The adult rat, rabbit or guinea pig may be used. Other
species may be used but their use would r uire justification.
(ii) . Adult animals should be used. The following weight ranges
at the start of the test are suggested in order to provide animals of a
size cthith facilitates the conduct of the test:
(A) Rats, 200 to 300 g.
(B) Rabbits, 2.0 to 3..Q kg.
(C) Guinea pigs, 350 to 450 g.
(iii) Sex.
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(A) Equal numbers of animals of each sex with healthy skin should be
used at each dose level.
(B) The females should be nulliparous and non—pregnant.
(iv) Numbers . (A) At least 20 animals (10 females and 10 males)
should be used at each dose level.
(B) If interim sacrifices are planned the number should be increased
by the number of animals scheduled to be sacrificed before c npletion of
the study.
(C) The number of animals at the termination of the study should be
adequate for a meaningful evaluation of toxic effects.
(2) Control groups . A concurrent control group is recanmended. This
group should be an untreated or sham treated control group or, if a vehicle
is used in administering the test substance, a vehicle control group. If
the toxic properties of the vehicle are not )eiown or cannot be made avail-
able, both untreated and vehicle control groups are recanmended.
(3) Satellite group . A satellite group of 20 animals (10 animals per
sex), if included, may be treated with the high dose level for 90 days and
observed for reversibility, persistence, or delayed occurrence, of toxic
effects for a post—treatment period of appropriate length, normally not
less than 28 days.
(4) Dose levels and dose selection . (i) In sub&ronic taxicity
tests, it is desirable to have a dose response relationship as well as a
no—observed—toxic effect level. Therefore, at least three dose levels
with a control and, where appropriate, a vehicle control (corresponding to
the concentration of vehicle at the highest exposure level) should be
used.
(ii) The highest dose level should result in toxic effect but not
produce severe skin irritation or an incidence of fatalities which would
prevent a meaningful evaluation.
(iii) The lowest dose level should not produce any evidence of toxicity.
where there is a usable estimation of human exposure the lowest level should
exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable effects. If sore than one intermediate dose is used the dose
levels should be spaced to produce a gradation of toxic effects.
(v) In the low and intermediate groups, and in the controls, the inci-
dence of fatalities should be low to permit a meaningful evaluation of the
results.
(5) Exposure conditions . The animals are treated with test substance,
ideally for at least 6 hours per day on a 7-days—per—week basis, for a
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period of 90 days. However, based primarily on practical considerations,
application on a 5—days—per—week basis is considered to be acceptable.
(6) Observation period . Duration of observation should be for at
least 90 days.
(7) Preparation of animal skin . (i) Shortly before testing, fur
is clipped from the dorsal area of the trunk of the test animals. Shaving
may be esployed, but it should be carried t approximately 24 hours before
the test. Repeat clipping or shaving is usually needed at approximately
weekly intervals. When clipping or shaving the fur, care must be taken to
avoid abrading the skin, which could alter its permeability.
(ii) Not less than 10 percent of the body surface area should be
clear for the application of the test substance. The weight of the animal
should be taken into acc t when deciding on the area to be cleared and on
the dimensions of the covering.
(iii) When testing solids, which may be pulverized if appropriate,
the test substance should be moistened sufficiently with water or, where
necessary, a suitable vehicle to ensure good contact with the skin. When a
vehicle is used, the influence of the vehicle on penetration of skin by the
tes t subs tance should be taken into ac cc*in t.
(8) Application of the test substance . (1) The test substance
should be applied unifonuly over an area which is approximately 10 percent
of the total body surface area. With highly toxic substances the surface
area covered may be less, but as much of the area should be covered with
as thin and uniform a film as possible.
(ii) During the exposure period the test substance is held in contact
with the skin with a porous gauze dressing and non—irritating tape. The
test site should be further covered in a suitable manner to retain the
gauze dressing and test substance and ensure that the animals cannot ingest
the test substance. Restrainers may be used to prevent the ingestion of
the test substance, but canpiete immobilization is not a recaiunended method.
(9) Observation of animals . Ci) A careful cageside examination
should be made at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of
weak or moribund animals, to ensure that not more than 10% of the animals
in any test group are lost iran the test due to cannibalism, analysis of
tissues, misplacement, and similar management problans.
(iii) Signs of toxicity should be recorded as they are observed,
including the time of onset, the degree, and duration.
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(iv) Cageside observations should include, but not be limited to,
changes in skin and fur, eyes and mucous membranes, respiratory, circulatory,
autonomic and central nervous system, somatornotor activity arid behavior
pattern.
(v) Animals should be weighed weekly. Food consumption data should
be collected weekly.
(vi) At the end of the study period all survivors in the non-satellite
treatment groups are sacrificed. Moribund animals should be removed and
sacrificed then noticed.
(10) Clinical examinations . (i) The following examinations should
be made on all animals of eath sex in each group:
(A) Hematology determinations should be investigated at the end of
the test period. Test areas cthieh are considered to be appropriate to all
studies are:
(! ) Hematocrit;
(2) Hemoglobin concentration;
(3) Erytbrocyte count;
(4) Total and differential leucocyte count; and
(5) A measure of clotting potential, suth as clotting time, prothrombin
time, thranboplastin time, or platelet count.
(B) Certain clinical biodtemistry determinations on blood should be
carried at the end of the test period. Test areas .hich are considered
appropriate to all studies are:
(1) Electroyte balance;
(2) Carbohydrate metabolism; and
(3) Liver and kidney function.
(C) The selection of specific tests will be influenced by observations
of the mode of action of the substance. Suggested determinations are:
.i) Calcium;
(2) Phosphorus;
( •) Chloride;
(4) Sodium;
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(5) Potassium;
(6) Fasting glucose (with period of fasting appropriate to the species);
(7) Serum glutamic—pyruvic transaminase (also known as serum alanine
axninotransferase);
(8) Serum glutamic—oxaloacetic transaminase (also known as serum
aspartite aminotransferase);
Urea nitrogen;
(10) Albumen;
(11) Blood creatinine;
(12) Total bilirubin; and
(13) Total serum protein meas mements.
(14) Other determinations thich may be necessary for an adquate toxi-
cological evalution include analyses of lipids, hormones, acid/base balance,
inethemoglobin, cholinesterase activity.
(15) Additional clinical bio±emistry may be employed, there necessary,
to extend the investigation of observed effects.
(ii) The following examinations should be made on all animals of each
sex in each group:
(A) Ophthalmological examination, using an ophthalmoscope or equiva-
lent suitable equipment. This examination should be made prior to exposure
to the test substance and at the termination of the study, preferably in
all animals but at least in the high dose and control groups. [ f &anges
in the eyes are detected, all animals should be examined.
(B) Urinalysis, but only when there is an indication of obtaining
useful data based on expected or obeerved toxicity.
(11) Gross necropsy . Ci) All animals shall be subjected to a full
gross necropsy which includes examination of:
(A) The external surface of the body,
(ii) The liver, kidneys, and testes should be weighed wet, as soon
as possible after dissection to avoid drying.
(12) Preservation of tissues . Ci) The following organs and tissues,
or representative samples thereof, should be preserved in a suitable medium
for possible future histopathological examination:
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(A) Normal and treated skin;
(B) All gross lesions;
(C) Brain - including sections of medulla/pons, cerebellar cortex and
cerebral cortex;
(D) Pituitary;
(E) Thyroid/parathyroid;
(F) Thymus;
(G) Lungs;
(H) Heart;
(I) Salivary glands;
(J) Liver;
(K) Spleen;
(L) Kidneys;
(M) Adrenals;
(N) Pancreas;
(0) Testes;
(P) Accessory genital organs, uterus;
(Q) Aorta;
(R) Gall bladder (if present);
(S) Esophagus;
(T) Stomach;
(U) Duodenum;
(V) Jejunum;
(W) Ileum;
(x) Caecum;
(Y) Colon;
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(Z) Rectum;
(AA) Urinary bladder;
(BB) Representative lymph node;
(CC) Peripheral nerve; and
(DD) Aorta.
(ii) The following tissues need be preserved only if indicated by signs
of toxicity or tar t organ involvement:
(A) Trachea;
(B) Sternum with bone marrow;
(C) Mammary gland;
(D) Thigh musculature;
(E) Eyes;
(F) Femur — including articular surface;
(G) Spinal cord at three levels — cervical, midthoracic, and lumbar; and
(H) Exorbita]. lachrymal glands.
(13) Histopathology . (1) The following histopathology should be
performed:
(A) Full histopathology on nozTnal and treated skin and on organs and
tissues, listed in paragraçhs (e)(12)(i) and (ii) of this section, of all
animals in the control and high dose groups.
(B) All gross lesions in all animals.
(C) Target organs in all animal groups.
(ii) The following histopathology should be performed:
(A) Lungs of animals (rats) in the low and intermediate dose groups
should be subjected to his topathologica]. examination for evidence of infec-
tion, since this provides a convenient assessment of the state of health
of the animals.
(B) When a satellite group is used, histopathology should be performed
on tissues and organs identified as showing effects in other treated groups.
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(h) Data and reporting . (1) Treatment of results . Ci) Data shall
be stinmarized in tabular form, showing for each test group the number of
animals at the start of the best, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each tYpe
of lesion.
(ii) All observed results, quantitative and incidental, shall be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical method should be selected
during the design of the study.
(2) Evaluation of results . The findings of a subthronic dermal toxi-
city study shall be evaluated in conjunction with the findings of preceding
studies and considered in terms of the observed toxic effects and the
necropsy and his topathological findings.
(i) The evaluation will include the relationship between the dose of
the test substance and the presence or absence, the incidence and severity,
of abnormalities, including:
(A) Behavioral abnormalities;
(B) Clinical abnormalities;
(C) Gross lesions;
(D) Identified target organs;
CE) Body weight changes;
(F) Effect on. mortality; and
(G) Any other general or specific toxic effects.
(ii) A properly conducted subchronic test should provide a satisfactory
estimation of a no—effect level.
(3) Test report . In addition o the information. r uired by 80-4,
the test report summary shall include the following information:
Ci) Toxic response and other effects data by sex and dose;
(ii) Species and strain; and
(iii) Individual animal data for the following:
(A) Time of death during the study or thether animals survived to
termination;
(B) Time of observation of each abnormal sign and its subsequent
course;
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(C) Body weigh t data;
CD) Hematological tests employed and results, with relevant baseline
data, if available;
(E) Clinical biodiemistry tests employed and results, with relevant
baseline data, if available;
(F) Necropsy findings;
(G) Detailed description of all histopathological findings; and
(iv) Statistical treatment of results, where appropriate.
§ 82—4 Subchronic inhalation toxicity: 90-day study .
(a) When required . Data from a subchronic inhalation study con-
ducted with the substance specified in paragraph Ce) are required by
40 CFR Part 158 to support the registration of each manufacturing—use product
and formulated product for which an acute inhalation toxicity determination
is required under §163.81—3, and whose pesticidal use may result in repeated
inhalation exposure.
Ci) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be suhnitted. Section 11—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who mus t s u hn.1. t the r equir ed data as a gener al rule.
(b) Purpose . The subdironic inhalation study is designed to determine
the non—observed effect level and toxic effects associated with continuous
or repeated exposure to a test substance for a period of 90 days. The
test is not capable of determining those effects that have a long latency
period for development (e.g., carcinogenicity and life shortening). It
provides information on health hazards likely to arise from repeated exposure
by the inhalation route over a limited period of time. It should provide
information on target organs, the possibilities of accumulation, and can
be of use in selecting dose levels of chronic studies and for establishing
safety criteria for human exposure. Hazards of inhaled substances are
influenced by the inherent toxicity arid by physical factors such as
volatility, particle size and exposure.
Cc) Definitions . (1) “Aerodynamic diameter” applies to the behavioral
size of particles of aerosols. It is the diameter of a sphere of *mit
density which behaves aerodynamically as the particle of the test substance.
It is used to c npare particles of different sizes, shapes and densities
to predict where in the respiratory tract such particles may be deposited.
This term is used in contrast to “optical,” “measured,” or “geometric
diameter” which is a representation of actual diameters which in themselves
cannot be related to deposition within the respiratory tract.
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(2) “Geometric mean diameter” or “median diameter” is the calculated
aerodynamic diameter which divides the particles of an aerosol in half
based on the weight of the particles. Fifty percent of the particles by
weight will be larger than the median diameter and 50 percent of the
particles will be smaller than the median diameter. The median diameter
and its geometric standard deviation is used to statistically describe the
particle size distribution of any aerosol based on the weight and size of
the particles.
(3) “Inhalable diameter” refers to that aerodynamic diameter of a
particle which is considered to be inhalable for the organism. It is used
to refer to particles which are capable of being inhaled and may be depos-
ited anywhere within the respiratory tract from the trachea to the deep
lung (the aveoli). For humans, inhalable diameter is considered here as
15 micrometers or less.
(4) “No—effect level”/”Non— toxic—effect level” /“No—advers e—effect
level”/”No—observed—effect level” is the maximum dose used in a test which
produces no observed adverse effects. It is expressed in terms of weight
or volume of the substance per urii t volume of air (mg/l or ppm).
(5) “Subthronic inhalation toxicity” is the adverse effects which
follow repeated daily exposure of experimental animals to a chemical by
inhalation for a significant part of an animal’s life span (usually less
than 10%).
Cd) Principle of the test method . Several groups of experimental
animals are exposed daily for defined periods (usually 6 hours per day,
5 days per week) to the test substance in graduated concentrations, one
concentration being used per group, for a period of 90 days. During the
period of administration the animals are observed daily to detect signs of
toxicity. Animals which die during the test are necropsied and at the
conclusion of the test surviving animals are sacrificed and necropsied and
appropriate histopathological examinations and laboratory analyses carried
out.
(e) Substance to be tested . The technical grade of the product
shall be tested. The chemical composition and physical state of the
substance being tested should, if pessible, be the same as that which is
encountered during the use of the product. Aerosol particles may have to
be reduced to sizes which are inhalable for the animal being tested
considering the entire respiratory system of the animal.
(f) Test procedures . (1) Animal selection . (i) Species and
strain . A variety of rodent species may be used although the rat is the
perferred species. If another mammalian species is used, the tester should
provide justification/reasoning for its selection. Commonly used laboratory
strains should be employed.
(ii) Young adult animals should be used • At the commencement
of the study the weight variation of animals should not exceed + 20 percent
of the mean weight for each sex.
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(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) Females should be nulliparous and non—pregnant.
(iv) Numbers . (A) At least 20 animals (10 female and 10 male)
should be used for each test group.
(B) If interim sacrifices are planned the number should be increased
by the number of animals scheduled to be sacrificed before the canpietion
of the study.
(C) The number of animals at the termination of the study must be
adequate for a meaningful evaluation of toxic effects.
(2) Control groups . One concurrent control group is reca1lmended. If
no vehicle is used, an air control group should be treated in the same
manner as all other test animals except this control group should not be
exposed to an atmosphere containing test substance. When a vehicle is used
to help generate the exposure atmosphere, the control group (vehicle control)
should be exposed to the greatest concentration of the vehicle used in the
study. Ideally, the vehicle should not significally affect the toxicological
response of the animals exposed to the test substance.
(3) Satellite group . A satellite group of 20 animals (10 animals
per sex), if included, may be treated with the high concentration level
and observed for reversibility, persistence, or delayed occurrence of
toxic effects for a post-treatment period of appropriate length, normally
not less than 28 days.
(4) Dose levels and dose selection . (1) In subdironic toxicity
tests, it is desirable to have a dose response relationship as well as a
no—observed—toxic—effect level • Therefore, at least three concentrations
with a control should be used.
(ii) The highest concentration should result in toxic effects but
not produce an incidence of fatalities thich would prevent a meaningful
evaluation.
(iii) The lowest concentration should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the lowest
concentration should exceed this.
(iv) Ideally, the intermediate dose level should produce minimal
observable toxic effects. If nore than one intermediate dose level is
used, the concentrations should be spaced to produce a gradation of toxic
effects.
(v) In the low and intermediate groups and in the controls the
incidence of fatalities should be low, in order to permit a meaningful
evaluation of the results.
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(vi) In the case of potentially explosive best substances care
should be taken to avoid generating explosive concentrations.
(5) Exposure durations . Animals should be exposed to the test
substance at least 6 hours per day 5 days per week over a 90 day period.
Longer or n re continuous exposures may be selected, depending on the test
substance and its expected use pattern. If shorter or less continuous
exposures seem appropriate, applicants should consult with the Agency
concerning the exposure times.
(6) Observation period . Ci) Duration of observation should be
for at least 90 days. (ii) Animals in a satellite group sdieduled for
follow-up observations should be kept for at least an additional 28 days
without treatment to detect recovery fr an, or persistence of, toxic effects.
(7) Inhalation equipment . (i) The animals should be tested in
inhalation equipment designed to substain a dynamic air flow of 12 to 15
air anges per hour, ensure an adequate oxygen content of a least 19 per
cent and an evenly distributed exposure atmosphere. ‘Jhere a thamber is
used its design should minimize crowding of the test animals and maximize
their exposure to the test substance. As a general rule, to ensure stability
of a thamber atmosphere, the total “volume t ’ of the test animals should not
exceed 5 percent of the volume of the test thamber. Maintenance of a
slight negative pressure inside the thamber will prevent leakage of the
test substance into surrounding area.
(ii) The temperature at which the test is performed should be
maintained at 22°C (+ 2°) for the rat. The relative humidity should be
maintained between 40 and 60 percent, unless the nature of the test sub-
stance or generating procedure (such as using water as a vehicle) precludes
this.
(iii) Alternatively, oro—nasal or head only exposures may be used
if animals exposed in chambers are excessively coated with test substance
and/or the whole body exposures produce high toxicity in the face or low
oral and dermal toxicity.
(8) Physical measurements . Measurements or monitoring should be
made of the following:
(i) The rate of air flow should be recorded at least every 60
minutes. Electronic mnitoring of air flow is desirable.
(ii) Actual concentrations of the test atmosçk ere fran the breathing
zone of the animals should be determined. Samples should be taken often
enough to th arac ten ze the a tmos e r e to wh ich the animals are exposed (a t
least one determination per run).
(iii) In the case of aerosols, particle size analyses of the exposure
atmospheres should be carried out as often as necessary to characterize the
aerosols to which the animals are exposed (at least once per run).
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(iv) chamber temperature should be recorded at least once every
60 minutes. Electron monitoring of temperature is desirable.
(9) Food and water . Food should be withheld during exposure and
water should not c ie in direct contact with the test atmospheres.
(10) Observation of animals .
(1) The animals should be observed clinically at least daily and
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation of weak or moribund
animals.
(ii) Signs of toxicity should be recorded as they are observed,
including the time of onset, the degree and duration.
(iii) Cageside observation should include, but not be limited to,
cthanges in the:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory;
CD) Circulatory;
CE) Autoncmtic and central nervous systems;
(F) Somatomotor activity; and
(G) Behavior pattern.
(v) Animals should be weighed weekly, and food consumption determined
weekly (if body weight is affected).
(vi) At the end of the posure period all survivors in the non—
satellite treatment groups are sacrificed. Moribund animals should be
removed and sacrificed then noticed.
(11) Clinical examinations . (1) The following examinations should
be made on at least 10 animals of eath sex in each group:
(A) Hematology determinations at a minimum should be carried ib at
the end of the test period. Determinations .kiid are considered to be
appropriate to all studies are:
I) Hematocrit;
(2) Hemoglobin concentration;
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(3) Erythrocyte count;
(4) Total and differential leucocyte count; and
(5) A measure of clotting potential, suth as clotting time, probhrombin
time, thrxtboplastin time, or platelet count.
(B) At a minimum, blood themistry determinations should be done
at the end of the test period. Test areas thich are considered appropriate
to all studies are electrolyte balance, carbohydrate metabolism, liver and
kidney function. The selection of specific tests will be influenced by
observations on the mode of action of the su1 tance. Suggested debermina—
Lions are:
1 ) Calcium;
(2) Ph phorus;
() Chloride;
(4) Sodium;
(5) Potassium;
(6) Fasting glucose (with period of fasting appropriate to the
species);
(7) Serum glutamic—pyruvic transminase (also known as serum alanine
aspartite axninotransferase);
(8) Urea nitrogen;
(9) P lbumen;
(10) Blood creatinine;
(11) Total bilirubin; and
(12) Total serum protein measurements.
(C) Other determinations thich may be necessary for an adquate toxi-
cological evaluation included analyses of lipids, hormones, acid/base
balance, methemoglobin, and cholinesterase activity.
(D) Additional clinical biochemistry may be employed, there necessary,
to extend the investigation of observed effects.
(ii) The following examinations should be made on at least 10 animals
of each sex in each group;
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(A) Ophthaln 1ogica]. examination, using an ophthalmoscope or
equivalent suitable equipment, should be made prior to exposure to the test
substance and at the termination of the study, preferably in all animals,
but at least in the high dose and control groups.
(B) Urinalysis on rodents are not suggested as routine procedures
but should be carried out when there is a need based on expected or observed
toxicity. An ng the parameters whith should be assessed are color, specific
gravity or osmolarity, pH, protein, glucose, ketones, formed elements
(RBC’s, WBC’s, epithelia]. cells, etc.), crystalline and anorphous materials,
and blood pigments.
(12) Gross pathology . (i) All animals should be subjected to a
full gross necropsy whid includes examinations of:
(A) The external, surface of the body;
(B) All orifices; and
(C) The cranial, thoracic and abdaninal cavities and their contents.
(ii) At least the liver, kidneys, lungs, and testes should be weighed
wet, as soon as possible after dissection to avoid drying.
(13) Tissue preservation .
(i) The following organs and tisst s, or representative samples
thereof, should be preserved in a suitable medium for possible future
his topa tho logical examination:
(A) Nasopharyngeal tissues;
(B) All gross lesions;
(C) Brain — including sections of medulla/pons, cerebella cortex,
and cerebral cortex;
(D) Pituitary;
(E) Thyroid/parathyroid;
(F) Thymus;
(G) Trachea;
(H) Lungs, which should be removed intact, weighed, and treated with
a suitable fi tive to ensure that lung structure is maintained (perfusion
Wi th the fixa tive is considered to be an effective procedure);
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(I) Heart;
(J) Salivary glands;
(K) Liver;
(L) Spleen;
CM) Kidneys;
(N) Adrenals;
(0) Pancreas;
(P) Testes;
CQ) Uterus;
CR) Aorta;
CS) Gall bladder (if present);
(T) Esophagus;
(U) Stomach;
CV) Duodenum;
(W) Jejunum;
(X) Ileum;
(Y) Caecuxn;
(Z) Colon;
(AA) Rectum;
(SB) Urinary bladder;
(CC) Representative lymph node;
(DD) Peripheral nerve;
(EE) Sternum wiU bone marrow; and
(FF) Eyes.
(ii) The following tissues need be preserved only if indicated by
signs of toxicity or targat organ involvement:
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(A) Skin;
(B) Accessory genital organs;
(C) Mammary gland;
(D) Thigh musculature;
(E) Femur — including articular surface;
(F) Spinal cord at three levels — cervical, midthoracic, and
lumbar; and
(G) Exorbital i.achryma]. glands.
(14) Histopathology .
(i) The following histopathology should be performed:
(A) Full histopathology on the respiratory tract and other organs
and tissues, listed in paragraph (f)(13) of this section, of all animals
in the control and high dose groups;
(B) All gross lesions in all animals; and
(C) Target organs in all animals.
(ii) The following histopathology should be performed: (A) Lungs
of animals in the low and intermediate dose groups should also be subjected
to histopathological ecamination, primarily for evidence of infection
since this provides a convenient assessment of the state of health of the
animals.
(B) en a satellite group is used, histopathology should be performed
on tissues and organs identified as showing effects in other treated groups.
(g) Data and reporting . (1) Treatment of results . (i) Data
shall be summarized in tabular form, showing, for ead test group:
(A) The number of animals at the start of the test;
(B) The number of animals showing lesions;
(C) The types of lesions; and
(D) The percentage of animals displaying eath type of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical method should be selected
during the design of the study.
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(2) Evaluation of results . The findings of a subdironic inhalation
toxicity study shall be evaluated in conjunction with the findings of
preceding studies and considered in terms of the observed toxic effects and
the necropsy and his topathological findings.
(i) The evaluation should include the relationship between the
concentration of the test substance and duration of exposure, and the
presence or absence, the incidence and severity, of abnormalities,
including:
(A) Behavioral abnormalities;
(B) Clinical abnormalities;
(C) Gross lesions;
(D) Identified target organs;
(E) Body weight dianges;
(F) Effects on n rtality; and
(G) Any other general or specific toxic effects.
(ii) A properly conducted 90—day subchronic test shall provide a
satisfactory estimation of a no—effect level.
(3) Test report . In addition to the information reccinmended by
§ 80-4, the test report summary shall include the following information:
(i) Test conditions . (A) Description of exposure apparatus,
including:
(1) Design;
(2) Type;
(3) Dimension;
(4) Source of air;
(5) System for generating particulates and aerosols;
(6) Method of conditioning air; and
(7) The method of housing animals in a test thaniber when this is
used.
(B) The uipment for measuring temperature, humidity and particulate
aerosol concentrations and size shall be described.
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(ii) Exposure data . These shall be tabulated and presented with
mean values and a measure of variability (e.g., standard deviation) and
shall include:
(A) Airflow rates through the inhalation equipment;
(B) Temperature and humidity of air;
(C) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air);
(D) Actual concentration in test breathing zone; and
(E) Particle size distribution (e.g., median aerodynamic diameter
of particles with deviation fran the mean).
(iii) Animal data . (A) Toxic response and other effects data by sex
and concentration;
(B) Species and strain used; and
(C) Individual animal data for the following:
(1) Time of death during the study or whether animals survived
to termination;
(2) Time of observation of each abnormal sign and its subsequent
course;
(3) Body weight data;
(4) Retnatological tests employed and all results;
(5) Clinical biodiemistry tests employed and all results;
(6) Necropsy findings;
(7) Detailed description and all histopathological findings; and
(D) Statistical treatment of results where appropriate.
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§ 82—5 Subchronic Delayed Neurotoxicity: _ 90—Day Study .
(a) When Required . As required by 40 CFR § 158.134, subthronic
delayed neurotoxici y studies shall be conducted if the acute delayed
neirotoxicity test showed n iropathy or n rotoxicity. See, specifically,
40 CFR § 158.50 and § 158.135 to determine whether these data must be
submitted. Section 11—A of this subdivision contains an additional discus-
sion of the “Formulators’ Exemption” and who must submit the required data
as a general rule.
(1) Definition .
“Subthronic delayed neirotoxicity” is a prolonged, delayed—onset
locomotor ataxia resulting from repeated daily administration of the
test substance.
(2) Controls .
If a positive control is used, a substance which is Iciown to produce
delayed neurotoxicity should be employed. Examples of suth substances are
tri-orthocresyl phosphate (T P) and leptophos.
(3) Principle of the Test Method .
Multiple dose levels of the test substance are administered orally
to domestic hens ( Gallus gallus domesticus ) for 90 days. The animals
are observed at least daily for behavioral abnormalities, locomotor
ataxia and paralysis. Histopathological examination of selected neural
tissues is undertaken at the termination of the test period.
(b) Description of the Test Procedure .
(1) Preparations .
Healthy young adult hens free fran interfering viral diseases and
medication and without abnormalities of gait should be acclimatized to the
laboratory coed i bio ns for the le as t five days prior to rand aniza tio n and
assignment to treatment and control groups.
(2) Experimental Animals .
(i) Selection of Species .
The adult domestic laying hen, aged between 8—14 n nths, is recaninended.
Standard size breeds and strains should be employed.
(ii) Number .
Ten hens should be used for each treatment and control group.
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(iii) Controls .
A concurrent control group should be used. This group should be
treated in a manner identical to the treated group, except that
administration of the test substance is omitted.
(iv) Rousing and Feeding Conditions .
Cages or enclosures which are large enough to permit mobility of the
hens and easy observation of gait should be used. Where the lighting is
artificial, the sequence should be 12 hours light, 12 hours dark.
Appropriate diets should be administered as well as an unlimited supply
of drinking water.
(3) Test Conditions .
Ci) Dose Levels .
At least three dose levels should be used in addition to the control
group(s). The highest dose level should result in toxic effects,
preferably delayed n irotoxicity, but not produce an incidence of
fatalities which would prevent a meaningful evaluation. The lowest dose
level should not produce any evidences of toxicity.
(ii) Route of Administration .
Oral dosing each day for at least five days per week should be carried
out, preferably by gavage or administration of gelatin capsules.
(4) Procedures .
The test or control substance should be administered and observations
begun. All hens should be carefully observed at least once daily throughout
the test period. Signs of toxicity should be recorded, including the time
of onset, degree and duration. Observations should include, but not be
limited to, behavioral abnormality, locomotor ataxia and paralysis. At
least once a week the hens should be taken outside the cages and subjected
to a period of forced motor activity, such as ladder climbing, in order to
enhance the observation of minimal responses. The hens should be weighed
weekly. Any moribund hens should be resoved and sacrificed.
(5) Pathology .
Ci) Gross necropsy .
Useful information is not usually provided by the results of gross
necropsy.
(ii) Histopathology .
Tissues from all animals should be fixed in situ , using whole
animal perfusion techniques. Sections should include medulla oblongata,
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spinal cord and peripheral nerves. The spinal cord sections should be
taken from the upper cervical bulb, the mid—thoracic and luinbo—sacral
regions. Sections of the proximal region of the tibial nerve and its
brandies and of the sciatic nerve should be taken. Unused portions
of the whole brain should be retained for possible future histopathology.
Sections should be stained with appropriate myelin and axon-specific
stains. Microscopic examination should be carried out on all hens in the
control and high-dose groups. Microscopic amination should also be
carried out on hens in the low and intermediate dose groups when there
is evidence of effects in the high-dose group.
(C) Data and Reporting .
(1) Treatment of Results .
Data may be summarized in tabular form, showing for each test group
the number of animals at the start of the test, the number of animals
showing lesions or effects, the types of lesions or effects, the percentage
of animals displaying eadi type of lesion or effect. All observed results
should be evaluated by statistical method if appropriate. A.ny generally
accepted statistical method may be used.
(2) Evaluation of Results .
The findings of a subthronic delayed ne.irotoxicity study should be
evaluated in conjunction with the findings of preceding studies and
considered in terms of the incidence and severity of observed ne.irotoxic
effects and any other observed effects and histopathological findings
in the treated and control groups. A properly conducted subthronic test
should provide a satisfactory estimation of a no—effect level based on
lack of clinical signs and histopathological dianges.
(3) Test Report .
The test r ort shall also include the following information:
(i) Doses administered (mg/kg);
(ii) Toxic response data by group with a description of dilnical
manifestations of nervous syst n damage where a grading system is used
the criteria should be defined;
(iii) Time of death during the study or whether animals survived
to termination;
(iv) The day of observation of each abnormal sign and its
subsequent course;
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Cv) Body weight data;
(vi) Necropsy findings for each animal, then performed;
(vii) A detailed description of all histopathological findings; and
(viii) Statistical treatment of results.
(4) Interpretation of Results .
This study provides information on the n irotoxic effects of repeated
exp ure to orgartophc phorous substances. Extrapolation from the results
of the study to man is valid only to a limited degree, although it can
provide useful information on the degree of neurotoxic activity of a
substance, no—effect levels and permissible human ecposure.
(5) Literature .
(1) M.B. AbQI—Donia, Ann. Rev. Pharmacol. Toxicol. 21 , 511-548 (1981)
(2) M.B. Abou—Donia and S.H. Pressig, Toxicol. Appi. Pharmocol. 38 ,
5995—6008 (1976).
(3) EPA—600/l—76—025 (edited by R.L. Baron) (National Te&n.
Info. Service, Springfield, VA, 1976).
(4) British Working Documents, October, 2, (Ministry of
Agriculture, Fisheries and Food, London, 1967).
(5) J.G. Cavanaugh, CRC Critical Reviews of Toxicology 2 (3),
465—417 (1973).
(6) F.R. Johannsen, P.L. Wright, D.E. Gordon, G.L. Levinskas,
R.W. Radue and P.R. Graham, Toxicol. Appi. Pharmacol. 41 ,
291—304 (1977).
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Series 83: CH1 DNIC AND LONG-TERM STUDIES
(NOTE: The sections of this series are prepared in conformity with the
guidelines developed by the Organization of Econanic Cooperation and
Development. Those guidelines were adapted to fit the toxicology data
requirements under FIFRA.]
§ 83—1 Chronic toxicity studies .
(a) When required . P .s required by 40 CFR § 158.135, chronic feeding
studies shall be conducted support the registration of each manufacturing-use
product and end—use product that meets either of the following criteria:
(1) Use of the pesticide product is likely to result in repeated
h tmtan cpos ur e to the pr, i ts active ingredient ( s) me tab oil tes, or
degradation products over a significant portion of the human life—span
(for example, products intended for use in and around residences, swimming
pools, and enclosed working spaces or their immediate vicinity); or
(2) The use requires a tolerance for the pesticide or an exemption
from the requirment to obtain a tolerance, or requires issuance of a food
additive regulation.
(3) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be suhnitted. Section Il—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must subsdt the required data as a general rule.
(b) Principle of Test Method . The objective of a chronic toxicity
study is to determine the effects of a substance in a mammalian species
following prolonged and repeated exposure. Under the conditions of this
test, effects which require a long latent period or are cumulative should
beccxne manifest. The application of these standards should generate data
on which to identify the majority of chronic effects and to determine
dose—response relationships. Ideally, the design and conduct should allow
for the detection of general toxicity including neurological, physiological,
biochemical, and hema to logical effects and exposure-rela ted morphological
(pathology) effects.
(b) Combined studies . For studies designed to meet the requirements
of both the chronic toxicity studies and the oncogenicity study, refer to
§ 83—5.
(d) Substance to be tested . Testing shall be performed with the
technical grade of each active ingredient in the product.
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(e) Test procedures . (1) Animal selection . (i) Species and strain .
Testing should be performed with two mammalian species, a rodent and a
non—rodent. The rat is the preferred rodent species and the dog is the
preferred non-rodent species. Ccinmonly-used laboratory strains should be
employed. If other mammalian species are used, the tester should provide
justification/reasoning for their selection.
(ii) (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are six, but in any case not more than
eight weeks old.
(B) Dosing of dogs should begin between four and six months of age
and in any case no later than nine months of age.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) The females should be nulliparous and non—pregnant.
(iv) Numbers . (A) For rodents, at least 40 animals (20 females and
20 males), and for non—rodents (dogs), at least eight animals (four females
and four males) should be used at each dose level.
(B) If interim sacrifices are planned the number should be increased
by the number of animals scheduled to be sacrificed before the canpletion
of the study.
(C) The number of animals iii any group should not fall below 50
percent at 15 imnths in mice and 18 months in rats. At the termination of
the experiment at 18 month in mice and 24 months in rats the survival in
any group should not fall below 25 percent.
(2) Control groups . (i) A concurrent control group should be uti-
lized. This group should be an untreated or sham treated control group
or, if a vehicle is used in administering the test substance, a vehicle
control group. If the toxic properties of the vehicle are not known or
cannot be made available, both nutreated and vehicle control groups are
recanmended.
(ii) In special circumstances such as in inhalation studies involving
aerosols or the use of an emulsifier of untharacterized biological activity
in oral studies, a concurrent negative control group should be utilized.
The negative control group is treated in the same manner as all other test
animals except that this control group should not be exposed to the test
substance or any vehicle.
(3) Dose levels and dose selection . Ci) In chronic toxicity tests,
it is desirable to have a dose response relationship as well as a no—observed—
toxic—effect level. Therefore, at least three dose levels should be used in
addition to the concurrent control group.
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(ii) The highest dose level in rodents should elicit some signs of
toxicity without causing excessive lethality; for non—rodents, there should
be no fatalities.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure, the lowest
dose level should exceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If n re than one intermediate dose is used, the
dose levels should be sraced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low to permit a meaningful
evaluation of the results. For non—rodents, there should be no fatalities.
(4) Observation period . The duration of the exposure period in rodents
for d emicals intended for a non—food use should usually be a least 12 rt nths
thile the duration of exposure in rodents for a food-use diemical should be
at least 24 nonths. The duration of exposure to non—rodents should be 12
months.
(5) Administration of the test substance . The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and theinical tharacteristics
of the test substance and the form typifying exposure in humans.
Ci) Oral studies . Provided it can be shown that the test substance
is absorbed from the gastrointestinal tract, the oral route of administra-
tion is preferred.
(A) The animals should receive the test substance in their diet, dis-
solved in drinking water, or given by gavage or capsule for a period of at
least 12 months.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed
five percent with the exception of nutrients.
CD) Ideally daily dosing on a 7—day per week basis should be used
because dosing in gavage or capsule studies on a 5-day per week basis may
permit recovery or withdrawal toxicity in the non-dosing period and thus
affect the result and subsequent evaluation. However, based primarily on
practical considerations, dosing on a 5-day per week basis is considered
to be acceptable.
(ii) Derinal studies . (A) The animals are treated by topical appli-
cation with the test substance, ideally for at least six hours per day.
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(B) The test substance should be applied uniformly over a shaved area
whid is approximately ten percent of the total body surfac area. With
highly toxic substances the surface area covered may be less, but as much
of the area should be covered with as thin and uniform a film as possible.
(C) Between applications, the test substance is held in contact with
the skin with a porous gauze dressing and non—irritating tape. The test
site should be further covered in a suitable manner to retain the gauze
dressing and test substance and ensure that the animals cannot ingest the
tee t substance.
(iii) Inhalation studies . (A) The animals are exposed by inhalation.
(B) The temperature at whid-t the test is performed should be maintained
at 22C ( . 2). Ideally, the relative humidity should be maintained between
40 to 60 percent, but in certain instances (e.g., tests of aerosols) this
may not be practicable. Food and water should be withheld during exposure.
(C) A dyn antic inh ala tb n sys tern wi th a sui table analytical concen tr a—
tion control system should be used. The rate of air flow should be adjusted
to ensure that condi tions throughout the equipment are essentially the same.
Maintenance of slight ne tive pressure inside the thamber will prevent
leakage of the test substance into the surrounding areas.
(6) Observation of animals . (i) A careful clinical examination should
be made at least once each week.
(iv) Daily cageside observations should include, but not be
limited to, thanges in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory;
(D) Circulatory;
(E) Autonanic and central nervous system;
(F) Somatomotor activity; and
(C) Behavior pattern.
(iii) Clinical signs of toxicity including suspected tumors and
mortailty should be recorded as they are observed, including the time of
Onset, the degree and duration.
(iv) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of
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weak or niz,ribund animals, to ensure that not irore than 10% of the animals
in any test group are lost fran the test due to cannibalism, analysis of
tissues, misplacement, and similar management problems.
(v) Body weights should be recorded individually for all animals once
a week during the first 13 weeks of the test period and at least once every
four weeks thereafter.
(vi) Measurements of food or water consumption should be determined
weekly during the first 13 weeks of the study and then at approximately
monthly intervals wdess health status or body weight dianges dictate other-
wise.
(vii) At the end of the study period all survivors are sacrificed.
Moribund animals should be removed and sacrificed when noticed.
(7) Physical measurements . For inhalation studies, measurements or
nonitoring should be made of the following:
(i) The rate of air flow should be monitored continuously and recorded
at intervals of at least once every 30 minutes.
(ii) During the exposure period the actual concentrations of the test
substance should be held as constant as practicable.
(iii) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol concen-
trations. During exposure, analysis should be conducted as often as
necessary to determine the consistency of particle size distribution.
(iv) Temperature and humidity should be monitc red continuously but
should be recorded at intervals of at least once every 30 minutes.
(8) Clinical pathology . (i) The following examinations should be
made on at least ten rats of ead sex per dose level and on all non—rodents:
(A) Hematology examinations (e.g., hemog).obin content, packed cell
volume, total red blood cells, total white blood cells, platelets, or
other measures of clotting potential) should be performed at approximately
six nonth intervals during the conduct of the study and at study termination,
on blood samples collected fran all non—rodents, and fran ten rats/sex of
all groups. If possible, these collections should be from the same rats
at each interval. If clinical observations sug st a deterioration in
health of the animals during the study, a differential blood count of the
affected animals should be performed. A differential blood count should
be performed on samples from those animals in the highest dosage group and
the controls. Differential blood counts are performed for the next lower
group(s) only if there is a major discrepancy between the highest group
and the controls. If hematological effects were noted in the subthronic
test, hematologica]. testing should be performed at 3, 6, 12, 18, and 24
months for a two—year study and at 3, 6, and 12 nonths for a one—year
study.
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(B) Certain clinical biocthemistry determinations on blood should be
carried out at least three times: at the beginning, middle, and at the
end of the test period. Blood samples should be drawn for clinical ±emistry
measurements from all non—rodents and at least ten rodents/sex of all groups,
if possible, frQn the same rodents at each time interval. Test areas
.thid are considered appropriate to all studies are electrolyte balance,
carbcthydrate metabolism, liver and kidney function. The selection of
specific tests will be influenced by observations on the mode of action of
the substance. Suggested determinations are:
(1) Calcium;
(2) Phosphorus;
(3) Chloride;
(4) Sodium;
(5) Potassium;
(6) Fasting glucose (with period of fasting appropriate to the species);
(7) Serum glutainic—pyruvic transaminase (also known as serum alanine
aminotrans ferase);
(8) Serum glutamic—oxaloacetie transaminase (also known as serum
aspartite aminotrans ferase);
(9) Blood urea nitrogen;
(10) Albumen;
(11) Blood creatinine;
(12) Creatine phosphokinase;
(13) Total diolesterol
(14) Total bilirubin; and
(15) Total serum protein measurements.
(16) Other determinations thich may be necessary for an adquate toxi-
cological evaluation include analyses of lipids, hormones, acid/base balance,
methemoglobin, and cholinesterase activi ty.
(17) Additional clinical biothemistry may be employed, ‘kere necessary,
to extend the investigetion of observed effects.
(ii) The following should be performed on at least ten rats of each
sex per dose level and all non—rodents:
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(A) Urine samples should be collected at the same intervals as in the
hematological examination des rThed in paragraph (e)(8)(i)(A) of this
section. The following determinations should be made fran either individual
animals or on a pooled sample/sex/group for rodents:
(1) Appearance;
(2) Volume;
(3) Specific gravity;
(4) Protein;
(5) Glucose;
(6) Ketones;
(7) Bilirubin;
(8) Occult bløod (semi—quantitatively); and
(9) Microscopy of sediment (semi—quantitatively).
(B) Ophthalmological examination, using an ophthalmoscope or equivalent
suitable equipment, should be made prior to the administration of the test
substance and at the termination of the study, preferably in all animals, but
at least in the high dose and control groups. If thanges in the eyes are
detected, all animals should be examined.
(10) Gross necropsy . Ci) A ccznplete gross examination should be done
in all animals, including those which died during the experiment or were
killed in moribund condition.
(ii) At least the liver, kidneys, brain and testes should be weighed
wet as soon as possible after dissection to avoid drying. For these organs,
at least ten rodents per sex per group and all non—rodents should be weighed.
(iii) If other clinical examinations are carried out, the information
obtained fran these procedures should be available before microscopic
examination, since they may provide significant guidance to the pathologist.
(11) Tissue preservation . The following organs and tissues, or
representative samples there of, should be preserved in a suitable medium
for possible future histopa tho logical examination:
(A) Skin;
(B) All gross lesions and tumors;
(C) Brain — including sections of medulla/pons, cerebellar cortex, and
cerebral cortex;
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(D) Pituitary;
(E) Thyroid/para thyroid;
(F) Thymus;
(G) Lungs/trachea;
(H) Heart;
(I) Sternum and/or femur with bone marrow;
(J) Salivary glands;
(K) Liver;
CL) Spleen;
(M) Kidneys;
(N) Adrenals;
(0) Pancreas;
(P) Testes;
(Q) Accessory genital organs, uterus;
CR) Female mammary gland;
(S) Musculature;
(T) Esophagus;
(U) Stomach;
(V) Duodenum;
(W) Jejunuin;
Cx) Ileum;
(Y) Caecum;
(Z) Colon;
(AA) Rectum;
(BE) Urinary bladder;
(CC) Lymph nodes;
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(DD) Peripheral nerve;
(EE) Spinal cord at three levels —— cervical, mi.dthoracic, and lumbar; and
(FF) Eyes;
(GG) Gall bladder; and
(HH) Aorta.
(ii) In special studies such as inhalation studies, the entire respiratory
tract should be studied, including nose, pharynx, and larynx.
(iii) Although inflation of lungs and urinary bladder with a fixative is
the optimal way to preserve these tissues, the inflation of the lungs in
inhalation studies is essential for appropriate histopathological examination.
(12) Histopathology . (i) The following histopathology should be performed:
(A) Full histopathology on the organs and tissues, listed in paragraph
(e)(ll) of this section, of:
(1) All non—rodents;
(2) All rodents in the control and high dose groups; and
(3) All rodents that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) The following histopathology should be performed:
(A) Lungs in all animals; special attention to examination of the
lungs of rodents should be made for evidence of infection since this provides
an assessment of the state of health of the animals;
(B) Liver in all animals; and
(C) Kidneys in all animals.
(iii) If excessive early deaths or other problems occur in the high
dose group ccznprcznising the significance of the data, the next dose level
should be examined for canplete histopathology.
(iv) In case the result of the experiment gives evidence of suhs tan—
tial alteration of the animals’ normal longevity or the induction of effects
that might affect a toxic respone, the next lower dose level should be
examined as described above.
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(v) An att npt should be made to correlate gross observations with
microscopic findings.
(C) Data and reporting . (1) Treatment of results . Ci) Data shall
be summarized in tabular form, showing for eath test group:
(A) The number of animals at the start of the test;
(B) The number of animals showing lesions;
(C) The types of lesions; and
CD) The percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical methods may be used; the statistical method should be selected
during the design of the study.
(2) Evaluation of study results . (A) The findings of a dtronic
toxicity study shall be evaluated in conjunction with the findings of
preceding studies and considered in terms of the toxic effects, the necropsy
and his topathological findings. The evaluation should include the relationship
between the dose of the test substance and the presence or absence, the
incidence and severity, of abnormalities, including:
! ) Behavioral abnormalities;
(2) Clinical abnormalities;
- (• ) Gross lesions;
(4) Identified target organs;
(5) Body weight thanges;
(6) Effects on mortality; and
(7) Any other general or specific or thronic toxic effects.
(B) In any study vthich demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailabilty of the
test substance should be considered.
(3) Test report . In addition to information r uired by 80-4, the
test report summary shall include the following information:
(i) Toxic response and other effects data by sex and dose;
(ii) Species, strain, and/or breed used;
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(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination;
(B) Time of observation of each abnormal sign and its subsequent course;
(C) Food or water consumption, if appropriate;
CD) Body weight data;
CE) Results of ophthalmol ical examination, when performed;
(F) Hematological tests employed and results, with relevant baseline
data, if available;
(G) Clinical biod emistry tests employed and results, with relevant
baseline data, if available;
(H) Necropsy findings;
(I) Detailed description of all histopathological findings; and
(iv) Statistical treatment of results, where appropriate.
§ 83—2 Oncogenicity study .
(a) When recjuired . Data frem oncogenicity testing are required by
40 CFR Part 158 to support the registration of each manufacturing-use product
and end—use product that meet any of the following criteria:
(1) The active in edient(s) or any of its (their) rnetabolites,
degradation products, or impurities:
Ci) Is structually related to a recognized carcinogen; or
(ii) Is a substance that causes mutagenic effect; or
(iii) Produces in subchronic studies a norphologic effect (e.g.,
hyperplasia, metap].asia) in any organ that may lead to neoplas tic cthange;
(iv) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be submitted. Section lI—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must submit the required data as a general rule.
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(2) The use requires a tolerance for the pesticide or exemption frxt
the requirement to obtain a tolerance, or requires the issuance of a food
additive regulation; or
(3) Use of the pesticide product is likely to result in human exposure
over a portion of the human life span whith is significant in terms of either
the time the exposure occurs or the duration of exposure (for example:
pesticides used in treated fabrios for wearing apparel, diapers, or bedding;
insect r ellents applied directly to human skin; pesticides applied to
tobacco; swimming pool additives; constant—release indoor pesticides which
are used in aerosol form).
(b) Purpose . The objective of a long—term carcinogenicity study is
to observe test animals for a major portion of their life span for the
development of neoplastic lesions during or after exposure to various doses
of a test substance by an appropriate route of administration.
(c) Combined studies . For studies designed to meet the requirements
of both the chronic toxicity studies and the oncogenicity study, refer to
83—5.
(d) Substance to be tested . Testing shall be performed with the
technical grade of eath active ingredient in the product.
(e) Test procedures . (1) Animal selection . (i) Species and strain .
It is recanmended that a ccmipound of unknown activity should be tested on
two mammalian species. Rats aiid mice are the species of choice because of
their relatively short life span, the limited cost of their maintenance,
their widespread use in pharmacological and toxicological studies, their
susceptibility to turior induction, and the availability of inbred or suffi-
ciently characterized strains. Cannonly used laboratory strains should be
employed. If other species are used, the tester should provide justification!
reasoning for their selection.
(ii) (A) Dosing of rodents should begin as soon as possible
after weaning, ideally before the animals are six, but in any case not riore
than eight weeks old.
(B) At ccxnmencement of the study, the weight variation of animals
should not exceed ± 20 percent of the mean weight for eath sex.
(C) Studies using prenatal or neonatal animals may be recanmended
under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) The females should be null.iparous and non—pregnant.
(iv) Numbers . (A) For rodents, at least 100 animals (50 females and
50 males) should be used at each dose level and concurrent control.
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(B) If interim sacrifices are planned the number should be increased
by the number of animals scheduled to be sacrificed before the canpletion
of the study.
(C) The number of animals in any group should not fall below 50
percent at 15 months in mice and 18 months in rats at the termination of
the experiment. At 18 month in mice and 24 months in rats the survival in
any group should not fall below 25 percent.
(2) Control groups . (i) A concurrent control group is required.
This group should be an untreated or sham treated control group or, if a
vehicle is used in administering the test substance, a vehicle control
group. If the toxic properties of the vehicle are not known or cannot be
made available, both mtreabed and vehicle control groups are recanmended.
(ii) In special circumstances such as in inhalation studies involving
aerosols or the use of an emulsifier of initharacterized biological activity
in oral studies, a concurrent negative control group should be utilized.
The’ negative control group is treated in the same manner as all other test
animals, except that this control group should not be exposed to the test
substance or any vehicle.
(3) Dose levels and dose selection . (i) For risk assessment purposes,
at least three dose levels should be used, in addition to the concurrent
control group.
(ii) The highest dose level should be sufficiently high to elicit
signs of minimal toxicity without substantially altering the normal life
s ça n.
(4) Exposure conditions . The animals are dosed with the test substance
ideally on a 7—days-per—week basis over a period of at least 24 months for
rats, and 18 months for mice. However, based primarily on practical consider-
ations, dosing on a 5—days—per—week basis is considered to be acceptable.
(5) Observation period . It is necessary that the duration of an
oncogenicity test ccinprise the majority of the normal life span of the
strain of animals to be used. This time period should not be less than 24
months for rats and 18 months for mice, and ordinarily not longer than 30
months for rats and 24 months for mice. For longer time periods, and where
any other species are used, consultation with the Agency in regard to the
duration of the test is advised.
(6) Administration of the test substance . The three main routes of
adminis tration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the p ) ysical and chemical characteristics of
the test substance and the form typifying exposure in humans.
(i) Oral studies . Provided it can be shown that the test substance
is absorbed from the gastrointestinal tract, the oral route of administration
is preferred.
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(A) The animals should receive the test substance in their diet,
dissolved in drinking water, or given by gavage or capsule for a period of
at least 24 n nths for rats and 18 nonthe for mice.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should generally
not exceed 5 percent with the exception of nutrients.
(ii) Dermal studies . (A) The animals are treated by topical
application with the test substance, ideally for at least 6 hours per day.
(B) The test substance should be applied uniformly over a shaved or
clipped area s t id is approximately ten percent of the total body surface
area. With highly toxic substances, the surface area co ,ered may be less,
but as muth of the area should be covered with as thin and uniform a film
as possible.
(C) During the exposure period the test substance may be held in
contact with the skin with a porous gauze dressing and nonirritating tape.
The test site should be further co rered in a suitable manner to retain the
gauze dressing and test substance and ensure that the animals cannot ingest
the test substance.
(iii) Inhalation studies . (A) The animals are exposed by inhalation.
(B) The temperature at s hid the test is performed should be maintained
at 22°C (±. 2°). Ideally, the relative hui idity should be maintained between
40 to 60 percent, but in certain instances (e.g., tests of aerosols) this
may not be practicable. Food and water should be withheld during exposure.
(C) A dynamic inhala n sys test wi th a sui table analytical concentration
control system should be used. The rate of air flow should be adjusted to
ensure that conditions throughout the equipment are essentially the same.
Maintenance of slight nec tive pressure inside the thamber will prevent
leakage of the test substance into the surrounding areas.
(7) Observation of animals . Ci) A careful clinical examination
should be made at least twice each week.
(ii) Daily cageside observations should include, but not be limited
to, changes in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory;
(D) Circulatory;
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(E) Autonomic and central nervous system;
(F) Somatomotor activity; and
(G) Behavior pattern.
(iii) Clinical signs and n rtality should be recorded for all animals.
Special attention should be paid to mass development. The following
information on each grossly visible or palpable mass should be recorded:
(A) Time of onset;
(B) Location;
(C) Size;
(D) Appearance; and
(E) Progression.
(iv) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead and isolation or sacrifice of
weak or noribund animals, to ensure that not irore than 10% of the animals
in any test group are lost fran the test due to cannibalism, autolysis of
tissues, misplacement, and similar management problems.
(v) Body weights should be recorded individually for all animals
once a week during the first 13 weeks of the test period and at least once
every four weeks thereafter.
(vi) Measurements of food or water consumption, respectively, should
be determined weekly during the first 13 weeks of the study and then at
approximately monthly intervals unless health status or body weight thanges
dictate otherwise.
(vii) At the end of the study period all survivors are sacrificed.
Moribund animals should be removed and sacrificed when noticed.
(8) Clinical pathology . At 12 nr,nths, 18 nznths, and at sacrifice,
a blood smear should be obtained fran 10 animals/sex/dosage group animals.
A differential blood count should be performed on blood smears fran those
animals in the highest dosage group ‘and the controls • If these data, or
data fran the pathological camination indicate a need, then the 12 and 18
nth blood smears fran other dose groups should also be examined. Differ-
ential blood counts are performed for the n t lower group(s) only if
there is a major discrepancy between the highest group and the controls.
If clinical observations suggest a deterioration in health of the animals
during the study, a differential blood count of the affected animals should
be performed.
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(9) Gross necropsy . (i) A complete gross examination should be
done in all animals, including those which died during the experiment or
were killed in moribund conditions.
(ii) At least the liver, kidneys, brain, and testes should be
weighed wet as soon as pass ib le after dis section to avoid drying. For
these organs, at least ten rodents rer sex group should be weighed.
(iii) If other dilnical examinations are carried out, the information
obtained from these procedures should be available before microscopic
examination, since they may provide significant guidance to the pathologist.
(10) Tissue preservation . The following organs and tissues, or
representative samples thereof, should be preserved in a s iitable medi.um
for possible future his topa tho logical examination:
(A) Masses and associated tissues of all animals;
(B) All gross lesions of all animals;
(C) Brain — including sections of inedulla/pons, cerebellar cortex,
and cerebral cortex;
CD) Pituitary;
CE) Thyroid/para thyroid;
(F) Thymus;
(G) Trachea;
(H) Lungs;
(I) Heart;
(J) Salivary glands;
(K) Liver;
CL) Spleen;
CM) Kidneys;
(N) Adrenals;
(0) Pancreas;
(P) Testes;
(Q) Accessory genital organs, uterus;
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(R) Female mammary gland;
(S) Skin;
CT) Esophagus;
(U) Stomach;
(V) Duodenum;
(W) Jejunum;
CX) Ileum;
(Y) Caecum;
(Z) Colon;
(AA) Rectum;
(BB) Urinary bladder;
(CC) Lymph nodes;
(DD) Peripheral nerve;
CEE) Spinal cord at three levels — cervical, midthoracic, and lumbar;
(FF) Sternum and/or f nur with bone marrow;
(GG) Musculature;
(HH) Eyes;
(II) Gall bladder; and
(JJ) Aorta;
(ii) In s cial studies such as inhalation studies, the entire
respiratory tract should be preserved, including nasal cavity, pharynx, and
larynx.
(11) Histopathology . (i) The following histopathology should be
performed:
(A) Full histopathology on organs and tissues, listed in paragraph
(e)(1) of this section, of all animals in the control and high dose groups
and all animals that died or were killed during the study;
(B) All gross lesions in all animals;
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(B) All gross lesions in all animals; and
(C) Target organs in all animals.
(ii) The following histopathology should be performed:
(A) Lungs in all animals (special attention to examination of the
lungs of rodents should be made for evidence of infection since this provides
an assessment of the state of health of the animals);
(B) Liver in all animals; and
(C) Kidneys in all animals.
(iii) If a significant difference is observed in hyperplastic, pre—
neoplastic or neoplastic lesions between the highest dose and control
groups, microscopic examination should be made on that particular organ or
tissue of all animals in the study.
(iv) If excessive early death or other problems occur in the high
dose group ccinprci ising the significance of the data, the next lower dose
level shall be examined for canpiete histopathology.
(v) In case the results of the experiment give evidence for substan-
tial alteration of the animals’ normal longevity or the induction of effects
that might affect a neoplastic response, the next lower dose level should
be examined as described above.
(vi) An attempt should be made to correlate gross observations with
microscopic findings.
(f) Data and reporting . (1) Treatment of results . (i) Data
shall be summarized in tabular form, showing, for eath test group:
(A) The n nber of animals at the start of the test;
(B) The number of animals showing lesions;
(C) The types of lesions; and
(D) The percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical methods may be used; the statistical method should be selected
during the design of the study.
(2) Evaluation of study results . (A) The findings of an oncogenic
toxicity study shall be evaluated in conjunction with the findings of
preceding studies and considered in terms of the toxic effects, the necropsy
and his topathological findings. The evaluation will include the relationship
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between the dose of the test substance and the presence or absence, the
incidence and severity, of abnormalities, including:
(1) Behavioral abnormalities;
(2) Clinical abnormalities;
Gross lesions;
(4) Identified target organs;
C) Body weight thanges;
(6) Effects on mortality; and
(7) Any other general or specific or toxic effects.
(B) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance shall be considered.
(3) Test report . In addition to information required by § 80-4, the
test report summary should include the following information:
Ci) Toxic response and other effects data by sex and dose;
(ii) Species and strain used; and
(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination;
(B) Time of observation of each mass and subsequent course;
(C) Food or water consumption, if appropriate;
(D) Body weight data;
(E) Hemato logical tests employed and results;
(F) Necropsy findings;
(G) Detailed description of all histopathological findings; and
(iv) Statistical treatment of results where appropriate.
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§ 83—3 Teratogenicity study .
( ) When required . Data on teratogenicity studies are required by
40 CFR Part 158 to support the registration of each manufacturing-use product
and end—use product which meets either of the following criteria:
(1) Its pesticidal use, under widespread and canit n1y recognized
practice, may reasonably be expected to result in significant exposure
of acute duration to human females; or
(2) Its use ‘requires a tolerance or an exemption fr i the requirement
to obtain a tolerance, or its use requires issuance of a food additive
regula tion.
(3) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be subsitted. Section lI—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must s uhul t the r equir ad data as a general rule.
(b) Purpose . The teratogenicity study is designed to determine the
potential of the test substance to induce structural and/or other abnormal-
ities to the fetus which may arise frem exposure of the mother during
pregnancy.
Cc) Definitions . (1) “Teratogenicity” is the property of a d-iemical
that causes permanent structural or functional abnormalities during the
period of embryonic development.
(d) Standard of the test method . The test substance is administered
in graduated doses, for at least that part of the pregnancy covering the
period of organogenesis, to several groups of pregnant experimental animals,
one dose level being used per group. Shortly before the expected date of
delivery, the pregnant females are sacrificed, the uteri removed, and the
contents examined for embryonic or fetal deaths, and live fetuses.
(e) Substance to be tested . Testing shall be performed with the
technical grade of eath active ingredient.
(f) Limit test . In the case of a substance of low toxicity, if a
dose level of at least 1000 mg/kg produces no evidence of embryo toxicity
or teratogenicity, studies at other dose levels may not be considered. If
a preliminary study at this high dose level, with definite evidence of
maternal toxicity, shows no adverse effects on embryos, studies at other
dose levels may not be considered necessary. Test procedures should follow
the guidelines of Section (g) below.
(g) Test procedures . (1) Animal selection . (i) Species and strain .
Testing should be performed in at least two mammalian species. The preferred
species are the rat and the rabbit. If other mammalian species are used, the
tester should provide justification/reasoning for their selection. Ccminonly
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used laboratory strains should be employed. The strain should not have low
fecundity and should preferably be diaracterized for its resjxnse to terato—
gens.
(ii) Young adult animals should be used.
(iii) Sex. Pregnant animals should be used at each dose level.
(iv) Number . At least 20 pregnant rats, mice or hamsters or 12 preg-
nant rabbits are recanmended at each dose level ar control group. The
objective is to ensure that sufficient pups are produced to permit meaning-
ful evaluation of the teratogeriic potential of the substance.
(2) Control group . A concurrent control group is recaumended. This
group should be an untreated or sham treated control group, or, if a vehicle
of i.mknown toxicity is used in administering the test substance, a vehicle
control group. Except for treatment with the test substance, animals in
the control group(s) should be handled in an identical manner to test
group animals.
(3) Dose levels and dose selection . (i) At least three dose levels
with a control or, a vehicle control as indicated in (2) above should be
used.
(ii) If a vehicle is used, its toxicological properties should be
understood; it should not be teratogenic nor have effects on reproduction.
(iii) To select the appropriate dose levels, a pilot or trial study
may be advisable. It is not always necessary to carry out a trial study in
pregnant animals. Comparison of the results frau a trial study in non-
pregnant, and the main study in pregnant animals will deironstrate if the
test substance is more toxic in pregnant animals.
(iv) Unless limited by the physical/cthemical nature or biological
properties of the substance, the highest dosage level should induce some
overt maternal toxicity sud as slight weight loss, but not rrore than 10
percent maternal deaths.
(v) The lowest dose level should not produce any evidence of tox-
icity.
(4) Observation period . Day 0 in the test is the day on which
vaginal plug and/or sperm are observed (where feas.thle). The dose period
should cover the period of major organogenesis. This may be taken as Days
6—15 for rat and mouse, 6—14 for hamster, or 6—18 for rabbit. If Day 0 is
based on observation of mating or artificial insemination, the times stated
should be adjusted by adding one day. Alternatively, the period of dosing
may be extended to approximately one day before the expected delivery
date.
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(5) Administration of test substance . Ci) The test substance or
vehicle is usually administered orally, by oral intubation, unless the
ehemical or physical tharacteristics of the test substance or pattern of
human cposure suggest a more appropriate route of administration.
(6) Exposure conditions . The female test animals are treated with
the tes t subs tance da il y throughout the appropriate tr tment pa nod • When
given by gavage, the dose may be based on the weight of the females at the
start of substance administration, or, alternatively, in view of the rapid
weight in whidi takes place during pregnancy, the animals may be weighed
periodically and the dosage based on the most recent weight determination.
(7) Observation of animals . (i) A careful examination for clinical
signs of toxicity should be made at least once each week to coincide with
weighing (vi).
(ii) Additional observations should be made daily, with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found dead, and isolation or sacrifice of
weak or n ribund animals, to ensure that not sore than 10% of the animals
in any test group are lost fran the test due to cannibalism, analysis
of tissues, misplacement and similar management problems.
(iii) Signs of toxicity should be recorded as they are observed,
including the time of onset, the degree, and the duration.
(iv) During the treatment and observation periods cageside observa-
tions should include, but not be limited to, dianges in:
(A) Skin and fur;
(B) Eye and mucous membranes;
(C) Respiratory system;
(D) Circulatory system;
(E) Autonanic and central nervous system;
(G) Sotnatomotor activity; and
(H) Behavioral pattern.
(v) Measurements should be made weekly of food consumption for those
animals in a dosed—feeding study.
(vi) Animals should be weighed weekly and at the time of sacrifice.
(vii) Females showing signs of abortion or premature delivery should
be sacrificed and subjected to thorough macroscopic emamination.
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(8) Gross necropsy . (i) At the time of sacrifice or death during
the study, the dam should be examined macroscopically for any structural ab-
normalities or pathological dianges which may have influenced the pregnancy.
(ii) Immediately after sacrifice or death, the uterus should be
removed and the contents examined for embryonic or fetal deaths and the
nunthe r of viable fe tuses • It is usually possible to es tima te the time of
death in utero where this has occurred.
(iii) The number of corpora lutea should be determined where possible.
(iv) The sex of the fetuses should be determined and each Utter
should be weighed, the weights recorded, and the mean fetal weight
derived.
(v) Following removal, each fetus should be examined externally.
(vi) For rats, mice and hamsters, one third to one—half of each Utter
should be prepared and examined for skeletal anomalies, and the remaining
part of each litter should be prepared and examined for soft tissue anomalies
using appropriate methods.
(vii) For rabbits, each fetus should be examined by careful dissection
for visceral anomalies and then examined for skeletal anomalies.
(h) Data and reporting . (1) Treatment of results . (i) Data shall
be summarized in tabular form, showing, for eath test group:
(A) The number of animals at the start of the test;
(B) The number of pregnant animals;
(C) The number of corpora lutea and resorptions (early and late);
(D) The number and percentages of live fetuses for each dose group
and in each litter; and
(E) The number of fetuses and litters containing fetuses with any
soft tissue or skeletal abnormalities.
(2) Evaluation of results . The findings of a teratogenicity study
shall be evaluated in terms of the observed effects and the dose levels
producing effects. It is necessary to cor ider the historical teratogenicity
data on the species/strain tested. Mc t properly conducted teratogenicity study
should provide a satisfactory estimation of a no—effect level.
(3) Test report . In addition to the information required by 80—4,
the test report shall include the following information:
(i) Toxic response data by dose;
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(ii) Species and strain;
(ii) Time of death during the study or whether animals survived to
termination;
(iii) Time of observation of each abnormal sign and its subsequent
course;
(iv) Food and body weight data;
(v) Pregnancy and Utter data; and
(vi) Fetal data (live/dead, sex, soft tissue and skeletal defects,
resorptions).
§ 83—4 Reproductive and fertility effects .
(a) When required . Data on a two—generation reproduction study are
required by 40 CFR Part 158 to support the registration of each manufacturing-use
product and formulated product that meets either of the following criteria:
(1) The pesticidal use of a pesticide product is likely to result in
significant human exposure to the product, its active ingredient(s),
metabolite(s), or degradation product(s); or
(2) The use requires a tolerance for the pesticide or an exemption
fran the requirement to obtain a tolerance, or requires issuance of a food
additive regulation.
(3) See, specifically, 40 CFR § 158.50 and §158.135 to determine
whether these data must be su nitted. Section 11-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must suhnit the required data as a general rule.
(b) Purpose . This guideline for two—generation reproduction testing
is designed to provide general information concerning the effects of a test
substance on gonadal function, estrus cycles, mating behavior, conception,
parturition, lactation, weaning, and the growth and development of the
offspring. The study may also provide information about the effects of the
test substance on neonatal n rbidity, nortality, and preliminary data on
teratogenesis and may serve as a guide for subsequent tests.
(C) Principle of the test method . The test substance is administered
to parental (P 1 ) animals prior to their mating, during the resultant preg-
nancies, and through the weaning of their F 1 offspring. The substance is
then administered to selected F 1 offspring during their growth into adult-
hood, mating, and production of an F 2 generation, up until the F 2 generation
is 21 days old.
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(d) Substance to be tested . Testing shall be performed with the
technical grade of each active ingredient in the product.
(e) Test procedures : (1) Animal selection . (i) Species and strain .
The rat or the mouse are the preferred species. If another mammalian species
is used, the tester should provide justification/reasoning for its selection.
Strains with low fecundity should not be used.
(ii) Parental (P 1 ) animals should be about 8 weeks old at the
start of dosing. See exposure conditior in paragraph (c)(4) of this
section for dosing regimen.
(i ii) Sex. (A) For an adequate assessment of fertility, both males
and females must be studied.
(B) The females should be nulliparous and non—pregnant.
(iv) Eath test and control group should contain at least 20 males
and a sufficient number of females to yield at least 20 pregnant females
at or near term.
(2) Control groups . (i) A concurrent control group is recanmended.
This group should be an untreated or sham treated control group or if a
vehicle is used in administering the test substance, a vehicle control
group.
(ii) If a vehicle is used in administering the test substance, the
control group should receive the vehicle in the highest volume used.
(iii) If a vehicle or other additive is used to facilitate dosing, it
should not interfere with absorption of the test substance or produce toxic
effects.
(3) Dose levels and dose selection . (i) At least three dose levels
and a concurrent control should be used.
(ii) The highest dose level should induce toxicity but not mortality
in the parental (P 1 ) animals.
(iii) The lowest dose level should not produce any evidence of toxi-
city. Where there is usable estimation of human exposure the lowest dose
should exceed this.
(iv) The intermediate dose level(s) should produce minimal observable
toxic effects. If nore than one intermediate dose is used, dose levels
should be spaced to produce a gradation of toxic effects.
(v) The incidence of fatalities in low and intermediate dose groups and
in the controls should be low, to permit meaningful evaluation of the results.
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(4) Exposure conditions . The animals are dosed with the test substance
ideally on a seven-days per week basis using the testing schedule presented
in Table 3.
Ci) Table 4 contains the dosing, mating, delivery, and sacrifice
schedule for animals on test.
(A) Daily dosing of the parental (P 1 ) males and females should begin
when they are about 8 weeks old. For both sexes, dosing should be continued
for at least eight weeks before the mating period.
(B) Dosing of P 1 males should continue through the mating period.
At the end of the mating period, P 1 males should be sacrificed and examined.
Dosing of the F 1 males saved for mating should continue from the time
they are weaned through the period they are mated with the F 1 females
(11 weeks for mice and 17 weeks for rats). F 1 males may be sacrificed
after the F 1 mating period.
(C) Daily dosing of the P 1 females should continue through the three
week mating period, pregnancy, and to the time of weaning three weeks
after delivery. Dosing of the F 1 females saved for mating should continue
fran the time they are weaned, through the period they are mated with the
F 1 males (11 weeks for mice and 17 weeks for rats), through pregnancy,
and to the weaning of the F 2 offspring.
(ii) All animals are sacrificed as follows:
(A) All P 1 males should be sacrificed immediately after delivery of
the litter last sired or, in cases of infertility, after the conditions of
(7)(i)(B) are met.
(B) F 1 males selected for mating should be sacrificed immediately
after delivery of the last F 1 litter sired, or, in cases of infertility, after
the conditions of (7)(i)(B) are met.
(C) F 1 males and females not selected for mating should be sacrificed
when weaned.
CD) The parental females in all generations should be sacrificed
upon weaning of their last litters.
CE) F 2 offspring are sacrificed when the offspring are 21 days of
age.
(5) Observation period . Duration of observation should be for at least
28 weeks fran dosing of P 1 animals to sacrifice of F 2 offspring at weaning.
(6) Administration of the test substance . The oral route of adm.this-
tration is preferred.
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Weeks
on
Study*
8—14
11—17
Dosing of P 1 males
may end at Week 25;
P 1 males are
sacrificed *
14—20 Dosing of P 1
females ends
P 1 females are
sacrificed
F 1 born and Utter
sizes randanly
adjusted to 8 pups
each
F 1 weaned; dosing
of F 1 females begins
F 1 offspring not
selected for mating
are sacrificed
22—34
F 1 mating; Dosing of
F 1 males may end at Week
40; F 1 males are
sacrif iced**
25—37
Remaining F 1 females
are sacrificed
F 2 born and
Utter sizes
randomly
adjusted to
to 8 pups each
F 2 offspring
are sacrf iced
*The lower number in the ranges is considered appropriate for mice, and the
upper number is appropriate for studies using rats. Earliest breeding age
for mice is approximately 8 weeks and that for rats is 14 weeks • This
schedule is for studies using one mating per generation. Numbers should be
adjusted by appropriate numbers of weeks to acccznmodate the second mating,
gestation and weaning of offspring as well as rest period between matings.
**This is appropriate for studies with two matings per generation or the
time may be longer in order that paragraph (7)(i)(A) can be satisfied.
TABLE 4. APPROXIMATE DOSING AND BREEDING SCHEDULE
P 1 F 1 F 2
0 Dosing of P 1 males
and females begins
P 1 mating period
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(1) When administered by gavage or capsule, the dosage administered
to eath animal prior to mating should be based on the individual animal’s
body weight and adjusted weekly. During pregnancy the dosage should be
based on the body weight at Day 0 and 6 of pregeancy.
(ii) It is recanmended that the test substance be administered in the
diet or drinking water.
(iii) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(iv) For a diet mixture, the highest concentration should not exceed
five percent with the exception of nutrients.
(7) Mating procedure . (i) Parental . (A) For each mating, each
female should be placed with a single randomly selected male from the same
dose level until pregnancy occurs or three weeks have elapsed. Paired
matings should be clearly identified and mixed matings with other males
avoided.
(B) Those pairs that fail to mate successfully should be evaluated
to determine the cause of the apparent infertility. This may involve such
procedures as additional opportunities to mate with other sires or dams,
histological examination of the reproductive organs, and examination of
the estrus or spermatogenic cycles.
(C) Each morning the female should be examined for presence of sperm
or vaginal plugs. Day 0 of pregaancy is defined as the day vaginal plugs
or sperm are found.
(ii) cross . (A) For mating the F 1 offspring, one male and one
female are randcrnly selected fran each litter for cross mating to produce
the F 2 generation. Mating of siblings should be avoided.
(B) F 1 males and females not selected for mating are sacrificed upon
weaning.
(iii) Special housing . Near parturition, pregnant animals should be
caged separately in delivery or maternity cages and provided with nesting
materials.
(iv) Standardization of litter sizes . (A) On Day 4 after birth, the
size of each litter should be adjusted by eliminating extra pups by randan
selection to yield, as nearly as possible, at least four males and four
females per litter.
(B) Whenever the number of male or female pups prevents having at least
four of eath sex per litter, partial adjustment (for example, five males
and three females) is permitted. Adjustments are not appropriate for
litters of less than eight pups.
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(C) Adjustments of the F 2 lit:ters is conducted in the same manner.
(8) Observation of animals . (i) A careful amination for c] inical
signs of toxicity should be made at least once each day. Pertinent behavi-
oral thanges, signs of difficult or prolonged parturition, food consumption
and all signs of toxicity, including n rtality, should be recorded. These
observations should be reported for each individual animal.
(ii) The duration of gestation should be calculated from Day 0 of
pregnancy.
(iii) Each litter should be examined as soon as pessible after deliv-
ery for the number of pups, stillbirths, live births, and the presence of
gross ancxnalies. Dead pups and pups sacrificed at Day 4 should be preserved
and studied for possible defects and cause of death. Live pups should be
counted and litters weighed, by weighing each individual pup (optional) at
birth, or soon thereafter, and on Days 4, 7 (optional), 14, and 21 after
birth.
(iv) Physical or behavioral abnorsalities observed in the dams or
offspring should be recorded.
(v) P 1 males and females should be weighed on the first day of dosing
and weekly thereafter. F 1 litters should be weighed at birth, or soon
thereafter, and on Days 4, 7, (optional) 14, and 21. Those F 1 offspring
selected to produce F 2 litters should be weighed at birth, or soon there-
after, and on days 4, 7 (optional), 14, and 21 after birth. Individual pup
weights should be measured at day 21.
(9) Gross necropsy . (i) A canpiete gross exarnina ion should be done
on all animals, including those ich died during the experiment or were
killed in moribund conditions, “to insure that not more than 10% of the
animals in any test group are lost from the test due to cannibalism,
analysis of tissues, misplacement and similar management problems.”
(ii) Special attention will be directed to the organs of the reproduc-
tive system.
(iii) The following organs and tissues, or representative samples
thereof, should be preserved in a suitable medium for possible future histo—
patho1 ical camination:
(iii) The following organs and tissues, or representative samples:
(A) Vagina;
(B) Uterus;
(C) Ovaries;
(D) Testes;
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CE) Epididymus;
(F) Seminal vesicles;
(G) Prostate; and
(H) Target organ(s) of all P 1 and F, animals selected for mating.
(10) Histopathology . (i) The following histopathology should be
performed:
(A) Full histopa tho logy on the organs listed in paragraph (e) (9) (iii)
of this section for all high dose and control P 1 and F 1 animals selected
for mating.
(B) Organs demonstrating pathology in these animals should then be
examined in animals from the other dose groups.
(C) Microscopic examination should be made of all tissues showing
gross pathological thanges.
(f) Data and reporting . (1) Treatment of results . (i) Data shall
be summarized in tabular form, showing, for eath test group:
(A) The number of animals at the start of the test;
(B) The number of animals pregnant;
(C) The types of change (see sections 83—4(e)(8),(9), and (10); and
(D) The percentage of animals displaying eacth type of thange.
(2) Evaluation of study results . An evaluation of test results,
including the statistical analysis, based on the clinical findings, the
gross necropsy findings, and the microscopic results, shall be made and
supplied. This should include an evaluation of the relationship, or lack
thereof, between the animal’s exposure to the test substance and the inci-
dence and severity of all abnormalities.
(3) Test report . In eddition to the information r uired by 80-4,
the test report shall include the following information:
Ci) Toxic response data by sex and dose, including fertility indices
and length of gestation;
(ii) Species and strain;
(iii) Time of death during the study or whether animals survived to
termina tion;
(iv) Toxic or other effects on r roduction, offspring, or postnatal
growth;
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Cv) Time of observation of each abnormal sign and its subsequent
course;
(vi) Body weight data for P 1 , F 1 and F 2 animals;
(vii) Necropsy findings;
(viii) Detailed description of all histopathological findings; and
(ix) Statistical treatment of results ere appropriate.
83—5 Combined chronic toxicity/oncogenicity studies .
(a) Purpose . The objective of a combined chronic toxicity/carcino—
genicity study is to determine the effects of a substance in a mammalian
species following prolonged and repeated exposure. The application of
these standards should generate data on which to identify the majority of
chronic and oncogenic effects and to determine dose—response relationships.
Ideally, the design and conduct should allow for the detection of neoplastic
effects and a determination of oncogenic potential as well as general toxi-
city, including neurological, physiological, biod-iemical, and hexnatological
effects and exposure-related n rphological (pathology) effects.
(b) Test procedures . (1) Animal selection . (i) Species and strain .
Preliminary studies providing data on acute, subchronic, and toxicokine tic
responses should have been carried it to permit an appropriate dioice of
animals (species and strain). As discussed in other guidelines, the mouse
and rat have been most widely used for assessment of carcinogenic potential,
while the rat and dog have been most often studied for chronic toxicity.
The rat is the species of choice for canbined thronic toxicity and carcino-
genicity studies. If other species are used, the tester should provide
justification/reasoning for their selection. Where available, the strain
selected should be susceptible to the carcinogenic or toxic effect of the
class of substances being tested if known, and provided it does not have a
spontaneous background too high for meaningful assessment. Cann nly used
laboratory strains should be employed.
(ii) . (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are six, but in any case not n re than
eight weeks old.
(B) At canmencexnent of the study, the weight variation of animals
should not exceed ± 20 percent of the mean weight for ead sex.
(C) Studies using prenatal or neonatal animals may be recanmended
under special conditions.
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(iii) Sex. (A) Equal numbers of animals of eath sex should be used
at each dose level.
(B) The females should be nulliparous and non—pregnant.
(iv) Numbers . (A) For rats, at least 100 animals (50 females and
50 males) should be used at each dose level and concurrent control.
(B) If interim sacrifices are planned the number should be increased
by the number of animals scheduled to be sacrificed before the canpletion
of the study.
(C) The number of animals in any group should not fall below 50 percent
at 15 months in mice and 18 months in rats. At the termination of the
experiment at 18 months in mice and 24 months in rats the survival in any
groups should not fall below 25 percent.
(2) Control groups . Ci) A concurrent control group (50 females and
50 males) should be utilized. These groups should be untreated or sham treated
control groups or, if a vehicle is used in administering the test substance,
vehicle control groups. If the toxic properties of the vehicle are not
known or cannot be made available, both untreated and vehicle control groups
are to be used. Animals (10/sex) in the satellite control group should be
sacrificed at the same time the satellite test group is terminated.
(3) Dose levels and dose selection . (i) For risk assessment purposes,
at least three dose levels should be used, in addition to the concurrent
control group.
(ii) The highest dose level in rodents should elicit signs of toxicity
without substantially altering the normal life span due to effects other
than tumors.
(iii) The lowest dose level should not produce any evidence of toxi-
city. Where there is a usable estimation of human expcsure the lowest
dose level should cceed this.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used the
dose levels should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low to permit a meaningful evalu-
ation of the results.
(vi) For thronic toxicological assessment, an additional treated and
a concurrent control satellite group may be included in the study. The
highest dose for satellite animals should be thosen so as to produce frank
toxicity, but not excessive lethality, in order to elucidate a toxicological
profile of the test substance.
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(4) Exposure conditions . Ideally the animals are dosed daily with
the test substance on a 7—days—per—week basis over a period of at least 24
months for rats and 18 months for nice and hamsters. The satellite animals
are dosed as above for at least a 12 month period, for a non-food use and
at least 24 months for a food use chemical.
(5) Observation period . It is necessary that the duration of the
oncogenicity test comprise the majority of the normal life span of the
animals to be used. It has been suggested that the duration of the study
should be for the entire lifetime of all animals. However, a few animals
may greatly exceed the average lifetime and the duration of the study may
be unnecessarily extended and complicate the conduct and evaluation of the
study. Rather, a finite period covering the majority of the expected life
span of the strain is preferred since the probability is high that, for the
great majority of chemicals, induced tumors will occur within such an
observation period. The following guidelines are recommended:
Ci) Generally, the termination of the study should be at 18 months
for nice and hamsters and 24 months for rats; however, for certain strains
of animals with greater longevity and/or low spontaneous tumor rate,
termination should be at 24 months for mice and hamsters and at 30 months
for rats. For longer time periods, and where any other species are used,
consultation with the Agency in regard to duration of the test is advised.
(ii) when included, the satellite groups and the concurrent satellite
control group should be retained in the study for at least 12 months for a
non—food use and at least 24 months for food use chemicals. These animals
should be scheduled for sacrifice for an estimation of test—substance—
related pathology uncomplicated by geriatric changes.
(6) Administration of the test substance . The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of adminstration depends upon the physical and chemical characteristics of
the test substance and the form typifying exposure in humans. The oral
route of adminis tration is preferred.
(i) For studies using oral administration, the following procedures
should be followed:
(A) The animals should receive the test substance in their diet,
dissolved in drinking water, or given by gavage or capsule for a period of
at least 24 months for rats and 18 months for mice and hamsters.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed
five percent, with the exception of nutrients.
(7) Observation of animals . (i) A careful clinical examination
should be made at least once each week.
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(ii) Daily cageside observations should include, but not be limited
to, changes in:
(A) Skin and fur;
(B) Eyes and mucous membranes;
(C) Respiratory;
(D) Circulatory;
(E) Auto nosic and centr a]. nervous sys tern;
(F) Somatomotor activity; and
(G) Behavior pattern.
(iii) Clinical signs of toxicity including suspected tumors and
mortality should be recorded as they are observed, including the time of
onset, the degree and duration.
(iv) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g., necropsy or
refrigeration of those animals found deed and isolation or sacrifice of
weak or moribund animals, to insure that not more than 10% of the animals
in any test group are lost fran the test due to cannibalism, autolysis of
tissues, misplacement, and similar management problems.
(i) Body weights should be recorded individually for all animals
once a week during the first 13 weeks of the test perlod and at least once
every four weeks thereafter.
(vi) Measurements of food or water consumption should be determined
weekly during the first 13 weeks of the study and then at approximately
monthly intervals unless health status or body weight thanges dictate
otherwise.
(vii) At the end of the study period all survivors are sacrificed.
Morthund animals should be removed and sacrificed when noticed.
(8) Clinical pathology . (i) The following examinations should be
made on at least 10 rodents of eath sex per dose level:
(A) Certain hematology determinations (e.g., hemoglobin content,
packed cell volume, total red blood cells, total white blood cells, plate-
lets, or other measures of clotting potential) should be performed at
approximately six month intervals during the conduct of the study and at
study termination, on blood samples collected fran 10 rodents/sex of all
groups. If possible these collections should be from the same animals at
each interval. Differential white blood cell counts of control and highest
dose animals, and only if necessary for the intermediate dose animals,
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should be determined at the same intervals. If clinical observations
suggest a deterioration in health of the animals during the study, a
differential blood count of the affected animals should be performed. A
differential leukocyte count should be performed on samples from those
animals in the highest dosage group and the controls. Differential
leukocyte counts are performed for the n ct lower group(s) only if there
is a major discrepancy between the highest group and the controls.
(B) Certain clinical biothemistry determinations on blood should be
carried out at approximately 6—nonth intervals and at termination. Blood
samples should be drawn fo r clinical th emi s try me as ur eme n t s Er an a t 1 e as t
ten rodents per s c of all groups and, if possible, from the same rodents
at each time interval. Test areas thich are considered appropriate all
studies are electrolyte balance, carbohydrate metabolism, and liver and kid-
ney function. The selection of specific tests should be influenced by obser—
vat ions on the node of action of the s ubs tance. Suggested determinations
are:
(!) Calcium;
(a) Phosphorus;
1) Chloride;
(4) Sodium;
(5) Potassium;
(6) Fasting glucose (with period of fasting appropriate the species);
(7) Serum glutaxnic-pyruvic transaminase (also known as serum alanine
aminotrans ferase);
(8) Serum glutatnic-oxaloacetic transaminase (also known as serum
aspartite aminotransferase);
(9) Blood urea nitrogen;
(10) Albumen;
(11) Creatine phosphokinase;
(12) Total cholesterol;
(13) Total bilirubin; and
(14) Total serum protein measurements.
(15) Other determinations thich may be necessary for an adequate
toxicological evalution include analyses of lipids, hornones, acid/base
balance, methemoglobin, and tholinesterase activity.
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(16) Additional clinical hiothemistry may be employed, there necessary,
to extend the investigation of observed effects.
(ii) The following should be performed on at least ten rodents of
eath sex per dose level:
(A) Urine samples, if possible fran the same rodents at the same
intervals as hematological examination above, should be collected for
analysis. The following determinations should be made fran either individ-
ual animals or on a pooled sample/sex/group for rodents:
(1) Appearance, volume, and specific gravity, for individual animals;
(2) Protein;
(3) Glucose;
(4) Ketones;
(5) Occult blood (semi—quantitatively); and
(6) Microscopy of sediment (semi—quantitatively).
(B) Ophthalmological camination, using an ophthalmoscope or equiva-
lent suitable equipment, should be made prior to the administration of the
test substance and at termination of the study, preferably in all animals,
but at least in the high dose and control groups. If thanges in the eyes
are detected all animals should be examined.
(9) Gross necropsy . (i) A canplete gross examination should be done
in all animals, including those which died during the experiment or were
killed in noribund conditions.
(ii) At least the liver, kidneys, brain, and testes should be
weighed wet as soon as possible after dissection to avoid drying. For
these organs, at least ten rodents per sex per group should be weighed.
(iii) If other clinical examinations are carried out, the information
obtained frc n these procedures should be available before microscopic
examination, since they may provide significant guidance to the pathologist.
(10) Tissue preservation . (i) The following organs and tissues, or
representative samples thereof, should be preserved in a suitable medium
for possible future histopathological examination:
(A) All gross lesions and tunors;
(B) Spinal cord at three levels — cervical, midthoracic, and lumbar;
(C) Brain — including sections of medulla/pens, cerebellar cortex and
cerebral cortex;
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(D) Pituitary;
(E) Thyroid/parathyroid;
(F) Thyinus;
(G) Trachea;
(H) Lungs;
(I) Heart;
(J) Salivary glands;
(K) Liver;
(L) Spleen;
(M) Kidneys;
(N) Adrenals;
(0) Pancreas;
(P) Testes;
(Q) Accessory genital organs, uterus;
(R) Female mammary gland;
(S) Gall bladder (if esent);
(T) Esophagus;
(U) Stomach;
(V) Duodenum;
(W) Jejunuin;
(X) Ileum;
(Y) Caecum;
(Z) Colon;
(AA) Rectum;
(BB) Urinary bladder;
(CC) Skin;
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(CC) Lymph nodes;
(DD) Peripheral nerve;
(EE) Sternum and/or fesur With bone marrow;
(FF) Musculature;
(GG) Eyes; and
(HH) Aorta.
(ii) In special studies, such as inhalation studies, the entire
respiratory tract should be preserved including nose, pharynx, and larynx.
(iii) Although inflation of lungs and urinary bladder with a fixative
is the optimal way to preserve these tissues, the inflation of the lungs only
in inhalation studies is essential for appropriate histopathological examin-
ation.
(11) Histopathology . (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tisst s, listed in paragraph
(b)(11) of this section, of all animals in the control and high dose groups
and all animals that died or were killed during the study;
(B) All gross lesions in all animals; and
(C) Target organs in all animals.
(ii) The following histopathology should be performed:
(A) Lungs of all animals (special attention to examination of the
lungs of rodents should be made for evidence of infection since this provides
an assessment of the state of health of the animals);
(B) Livers of all animals; and
(C) Kidneys of all animals.
(iii) If excessive early deaths or other problems occur in the high
dose group ccznpranising the significance of the data, the next dose level
should be examined for canplete histopathology.
(iv) In case the result of the experiment gives evidence of substan-
tial alteration of the animals’ normal longevity or the induction of effects
that might affect a toxic resrxnse, the next lower dose level should be
examined as described above.
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(v) An att npt should be made to correlate gross observations with
microscopic findings.
(c) Data and reporting . (1) Treatment of results . (i) Data
shall be summarized in tabular form, showing for each test group:
(A) The number of animals at the start of the test;
(B) The number of animals showing lesions;
(C) The types of lesions; arid
CD) The percentage of animals displaying each type of lesion.
(ii) All observed results, quantitative and incidental, should be
evaluated by an appropriate statistical method. Any generally accepted
statistical methods may be used; the statistical method should be selected
during the design of the study.
(2) Evaluation of study results . (A) The findings of a canbined
chronic toxicity/oncogenicity study shall be evaluated in conjunction with
the findings of preceding studies and considered in terms of the toxic
effects, the necropsy and histopathological findings. The evaluation should
include the relationship between the dose of the test substance and the
presence or absence, the incidence and severity, of abnormalities, including:
(1) Behavioral abnormalities;
(2) Clinical abnormalities;
2.) Gross lesions;
(4) Identified target organs;
(5) Body weight changes;
(6) Effects on nxrbality; and
(7) Any other general or specific or chronic toxic effects.
(B) In any study which deu nstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered. In order for a negative test to be
acceptable, it should meet the following criteria:
(3) Test report . In addition to information required by 80-4, the
test report summary shalfInclude the following information:
Ci) Toxic respense and other effects data by sex arid dose;
(ii) Species and strain used;
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(iii) Individual animal data for the following:
(A) Time of death during the study or whether animals survived to
termination;
(B) Time of observation of each abnormal sign and its subsequent
course;
(C) Food or water consumption;
(D) Body weight data;
(E) Results of ophthalmological camination, when performed;
(F) Hematological tests employed and results, with relevant baseline
data, if available;
(G) Clinical biodiemistry tests employed and results, with relevant
baseline data, if available;
(H) Necropsy findings;
(I) Detailed description of all histopathological findings; and
(iv) Statistical treatment of results where appropriate.
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Series 84: Mutagenicity.
84— 1 Purpose and General Recommendations for Mutagenicity Testing .
(a) When required . As required by 40 CFR § 158.135 mutagenicity data
shall be submitted to support the registration of each manufacturing—use
product and end—use product that meet any of the following criteria:
(1) The use requires a tolerance for the pesticide or exemption
from the requirement to obtain a tolerance, or r uire the issuance of
a food additive regulation; or
(2) The pesticide product is likely to result in significant
human exposure; or
(3) The active ingredient(s) or any of its (their) metabolites is
structurally—related to a mutagen or oncogen, or belongs to any chemical
class of compounds containing a significant number of mutagens or oncogens.
(4) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be submitted. Section 11—A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and who
must submit the required data as a general rule.
(b) Purpose . For each test substance a battery of tests is required
to assess the potential to affect the qualitative or quantitative integrity
of the mammalian cell’s genetic components. The objectives underlying the
selection of a battery of tests for mutagenicity assessment are:
(1) To detect, with sensitive assay methods, the capacity of a chemical
to alter genetic material in cells;
(2) To determine the relevance of these mutagenic changes to mammals,
and when mutagenic potential is demonstrated;
(3) To incorporate these findings in the assessment of heritable
effects, oncogenicity, and possibly, other health endpoints.
(c) Substance to be tested . Testing shall be performed with the
technical grade of each active ingredient in the product.
(d) Standards for metabolic activation . (1) Chemicals are often non—
mutagenic unless converted to an active mutagen by metabolic processing. The
reverse can also occur. Therefore, a metabolic activation system should be
incorporated into any test system other than intact mammals and insects.
(2) The test substance should be tested both in the presence and the
absence of mammalian tissue extracts (with appropriate cofactors) which
have been demonstrated to convert a wide range of chemical “proniutagens”
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(substances which are mutagenically—inactive in the absence of the tissue
extracts) to mutagenically—active substances. Rat liver extracts are
preferred. The tissue should be pre—induced for the relevant enzymatic
activities when appropriate. The inducer should be effective for the
class of compounds under test. Other tissue extracts should be used in
addition to Liver extracts when the principal site of metabolism of th
test substance is known not to be the liver, or when other tissues, includ-
ing plant tissue, are known to give positive results with chemicals struc-
turally—related to these chemicals.
(3) The test substance may also be exposed to metabolic processing in
intact mammals by a host—mediated system in which the target cells are in-
serted into host tissues or body cavities. Hepatocytes may also be used
to provide metabolic processing, either as a co—culture with a target cell,
or as the primary assay system.
Ce) Controls . all assays should be run with concurrent positive and
negative controls with the possible exception of the mouse specific locus
test.
(1) Positive controls . Positive control compounds should be selected
to demonstrate both the sensitivity of the indicator organism and the
functioning of the metabolic activation system. Positive controls should
also be selected to demonstrate the sensitivity of the indicator cells or
organisms to a compound with chemical characteristics similar to those of
the test substance. For instance, an alkylabing agent should be used as a
control for an expected alkylator, and art intercalating agent for a sus cted
intercalator. Where applicable, the positive control should be administered
by the same route as the test subs tance.
(2) Negative controls . Both a solvent and where applicable, a
nonsolvent negative control, should also be included.
84—2 Mutagenicity Tests .
(a) Tests required . The battery must include tests appropriate to
address the following three cate ries in accordance with the purposes
(84—1(b)):
(1) Gene mutations;
(2) Structural chromosomal aberrations; and
(3) Other genotoxic effects as appropriate for the test substance,
e.g., numerical chromosome aberations, direct DNA damage and repair.
(b) Representative tests . A representative selection of tests within
each category follows. The most commonly used organism, cell type, or
animal is indicated; others may be acceptable if sufficient testing is done
to verify their usefulness.
(1) Gene mutation tests .
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(1) Microorganisms.
( ) Bacteria, reverse mutation:
Salmonella typhimurium , Ames’ strains
Escherichia coli , WP2 and WP2 uvrA
Bacillus subtilis TKJ 5211, TKJ 6321
(B) Eucaryotic microorganisms, forward and reverse mutations:
Saccharomyces cerevisiae
Schizosaccharomyces ponibe
Neurospora crassa
Aspergillus nidulans
(ii) Suhuammalian organisms, sex—linked recessive lethal:
Drosophila melanogas ter
(iii) Mammalian cells in culture, forward or reverse mutations at
specific foci:
Chin e hamster lung (V79)
Chinese hamster ovary (do)
Mouse Lymphoma (L5178Y)
(iv) Specific locus:
Mouse
(2) Structural chromosome aberration tests .
(i) Eucaryotic microorganisms:
Aspergillus nidulans
Neurospora crassa
(ii) Suhnamxnalian organisms, chrcinosome tests:
Drosophila melanogaster
(iii) Mammalian cells in culture:
(A) Sister chranatid exthange
(B) Cytogenetic analysis
(iv) Mammals:
(A) Micronucleus tes t
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(B) Sister chromatid excthange
(C) Cytogenetic analysis
CD) Dominant lethal:
Mouse
Pa t
CE) Heritable translocation:
Mouse
(3) Tests for other genotoxic effects .
Ci) DNA dama arvi repair.
(A) Differential toxicity in bacteria:
Escherichia coli pc i A+/pol A—
Bacillus subtilis H17/M45
(B) Mitotic recombination in eucaryotic organisms:
Saccharomyces cerevisiae
Aspergillus nidulans
(C) Unscheduled DNA synthesis:
Mammalian cells in culture
Mouse
CD) DNA alkaline elutiort:
Cells in culture
CE) Sister diranatid cthange:
Cells in culture
Mammals
(ii) Numerical thromosomal aberrations.
(A) Eucaryotic microorganisms, mitotic segregation.
(B) Mitotic interference:
Cells in culture
Mammals
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(C) Micronucleus test:
Cells in culture
Mammals
(iii) Mammalian cell transformation:
Cells in culture
(iv) Target organ/cell analysis:
(A) Sperm norphology
(B) DNA synthesis inhibition
(C) DNA alkylation
(c) Current standards for test protocols, conduct of study arid preseriba—
tion of data are found in publications from the Gerie—Tox Program of the
EPA Office of Toxic Substances (appearing in Mutation Researth) and the
EPA/SRI International Project “ In Vitro Mutagenicity Studies of Environmen-
tal ( emicals,” 1982. Improvements in state of the art criteria will be
listel by EPA in the NTIS.
Because of the rapid improvements in this field, registrants are
encouraged to discuss with the Agency testing battery selection, protocol
design, and results of preliminary testing.
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Series 85: SPECIAL STUDIES
[ NOTE: Section 85—1 of this series is prepared in conformity with the
guideU.nes developed by the Organization of Econcmic Cooperation and
Development. Those guidelines were adapted to fit the toxicology data
requirements of this section under FIFRA.
Section 85—2 is essentially identical to § 163.86—1 of the Subpart F
guidelines proposed on August 22, 1978 (43 FR 37336). Public canments on
this section have not yet been used to revise the section. When the revision
is prepared, it will replace the current § 85—2.]
§ 85—1 Metabolism study .
(a) When required . Data fran a general metabolism study are required
to support the registration of each manufacturing—use product and eacth end-
use product which requires a thronic toxicity study or an oncogenicity
study, in accordance with 40 CFR § 158.135.
(1) See, specifically, 40 CFR § 158.50 and § 158.135 to determine
whether these data must be su xiitted. Section Il-A of this subdivision
contains an additional discussion of the “Formulators’ Exemption” and
who must su1 nit the required data as a general rule.
(b) Purpose . (1) Data fran studies on the absorption, distribution,
excre t ion, and metabolism of a tes t di exnic al are desirable to a id in the
evaluation of test results fran other toxicology studies and in the extra-
polation of data from animals to man. Suth studies should ideally be done
on each chemical of toxicological concern. The concern may be predicated
on the level and type of toxicity observed (or anticipated) and by the
magnitude of potential human exposure to the dieinical. Flexibility is
needed in the conduct of metabolism studies and depends on the tharacteris-
tics of the test themical being investigated. The main purpose of metabo-
lism studies is to produce data whith fortify the understanding of the
safety of the themical in consideration of its intended uses and antici-
pated human exposure.
(2) In addition to the general reasons stated in paragraph (b)(l) of
this section, a metabolism study may be performed for the following purposes:
(1) To determine the amount and rate of absorption of the test themical
at different dose levels;
(ii) To determine the pattern of distribution of the test themical
anong tissues, organs, and fluid canpartments at different dose levels,
after single and repeated dosages;
(iii) To identify and, to the extent possible, quantify significant
metabolites;
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(iv) To characterize route(s) and rate(s) of excretion;
(v) To determine any possible bioaccumulation and/or bioretention of
the test substance and/or metaboiltes; and
(vi) To determine absorption, metabolism, excretion, and distribution
as a function of single or repeated doses. For certain chemicals, metabo-
lism studies may not adequately define all of these processes.
(C) Labeled test material . (1) Single—dose testing shall be per-
formed with an analytically pure grade of the active ingredient, usually
in an isotopically—labeled form. The label may be radioactive, such as
l4 , 35 s, and 36 C1, or stable, such as 15 N and 180. In some cases, more
than one label per molecule may be advantageous. Labels shall be placed
in positions that may be expected to follow the ‘core” of the molecule or
significant portions thereof. If possible, one should avoid placing 14 C
in positions from sthith it may be expected to enter the carbon pool of the
test animal. Use of readily exchangeable labeling shall be avoided.
(2) Labeled compound may not be required if sufficiently selective
and sensitive physical—chemical tests for identifying the compound and its
metabolites are used.
(3) Some animals are to receive repetitive doses of nonlabeled
chemical substance (analytical grade).
(d) Test procedure . (1) Choice of method . A registrant may, after
consultation with the Agency, utilize a modified or completely different
experimental design if it provides the information required by this section.
(2) Animal selection . (i) Species and strain . The preferred
species is the rat. If another mammalian species is used, the tester
should provide justification/reasoning for its selection. Commonly—used
laboratory strains should be employed. Preliminary studies may be performed
in several species to develop information on comparative metabolism.
Information derived from preliminary studies may help in the selection of
species for subsequent toxicity tests.
(ii) . Young adult animals should be used. For specific pur-
poses, a comparative study using very young or very old animals may provide
information about the effects of age on the metabolism.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) Females should be riulliparous and nonpregnant.
(iv) Numbers • At least ten animals (five females and five males)
should be used at each dose level.
(3) Dose levels and dose selection . (i) At least two dose levels
should be used.
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(ii) The low dose should correspond to a no—effect level.
(iii) The upper dose should produce toxic or pharmacologic signs,
but should not produce severe effects or a high incidence of n rtality
which would prevent a meaningful evaluation.
(iv) The determination of absorption, tissue distribution, and
elimination should be studied as a f .mction of single or repeated doses.
(4) Observation period . P nimals should be kept in individual
metabolism cages for 7 days after radioactive dose or until 90+ percent of
the administered dose is excreted (whichever occurs first), at which time
all of the animals should be killed.
(5) Administration of the test substance . Ci) Route of adminis-
tration . The study should be done using the oral route (capsule or gavage).
If another route of adminis ation is used, the tester should provide
justification/reasoning for its selection. When vehicles are used, atten-
tion must be given to the possibility that they may interfere with the
kinetics of the test chemical.
(ii) Animal groups . The following four groups of animals should be
studied:
(A) Group A . These animals should each receive a single intravenous
dose of the labeled test substance at the low dose. If it is not possible
to dissolve the test Substance in physiological saline or water, this group
may be omitted.
(B) Group B . These animals should each receive a single oral dose
(by capsule or intubation) of the labeled test substance at the low dose.
(C) Group C . These animals should each receive a series of single
daily oral doses of the nonlabeled test substance (by capsule or intuba—
tion) over a period of least 14 days, followed at 24 hours after the last
dose by a single oral dose (by capsule or intubation) of the labeled
test substance. Each dose should be the low dose level.
(D) Group D . These animals should each receive a single oral
dose (by capsule or intubation) of the labeled test substance at the high
dose level.
(6) Observations of animals . (i) Distribution . For all animals
in Groups B, C, and D, the quantity of label in tissues and organs should
be measured at sacrifice by suitable methods with particular attention to
bone, brain, fat, nads, heart, kidney, liver, lungs, muscle, spleen,
tissues which displayed pathology (in this or prior studies), and residual
carcass.
(ii) Metabolism . Urine and feces fran all groups should be analyzed
by suitable methods in order to determine the extent of absorption and
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biotransformation and to identify the metabolites. An assay method for
detection of each major metabolite may be requested by the Agency.
(iii) Excretion . Quantities of label in urine, feces, and expired
air should be measured at appropriate intervals (e.g., 4, 8, 12, and 24
hours, 1.5, 2, 3, 4, 5, 6, and 7 days) throughout the study for all
animals. However, if a preliminary study shows no volatile label
materials are exhaled during the period of zero to 24 hours after dosing,
such evidence may be submitted in lieu of measuring label in the expired
air for this study.
(e) Data and reporting .
(1) Treatment of results . Data shall be summarized in tabular
form.
(2) Evaluation of results . Results, .here appropriate, shall be
evaluated statistically.
(3) Test report . In addition to the information required by 80—4,
the test report shall include the following data derived from tests on
animals in all groups:
(1) Quantity of isotype, together with rcent recovery of the
administered dose, in feces, urine, and the following tissues and organs of
animals in all groups:
(A) Bone;
(B) Brain;
(C) Fat;
(D) Testes;
CE) Heart;
(F) Kidney;
(G) Liver;
(H) Lung;
(I) Blood;
(J) Muscle;
(K) Spleen;
(L) Tissues which displayed pathology (in this or prior studies);
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(M) Literus; and
(N) Residual carcass.
(ii) Percent absorption; if possible, percent absorption by the oral
route in Groups B, C, and D;
(iii) A full description of the sensitivity and precision of all
procedures used to produce the data;
(iv) Information on the degree (i.e., specific activity for a
radiolabel) and site(s) of labeling of the test substance; and
(v) Counting efficacy data; such data should be recommended,
however, only upon specific request of the Agency.
(f) Additional metabolism studies . Additional, more specific
studies may be required to clarify important points.
(1) Some areas for possible further study include:
(i) Identification of tissue residues;
(ii) Binding by macromolecules in the blood, liver, gonads, and
other tissues; plasma binding studies may be conducted, usually in
vitro with plasma;
(iii) Placental transfer; placental transfer of a d emical sub-
stance may be determined by dosing pregnant rodents with themicals and
assaying their fetuses for the theinical;
(iv) Entrance into breast milk;
(v) Biotransformation by specific organs, tissues, and cell
fractions; and
(vi) Absorption by dermal or inhalation routes of exposure.
(2) Additional species may be utilized, since the rat and dog
differ significantly in metabolic pattern.
§ 85—2 Domestic animal safety testing .
(a) When required . Data from tests on domestic animals may be
required in accordance with 40 CFR § 158.135 to support the registration
of an end—use product if cats, dogs, cattle, pigs, sheep, or other danesti-
cated animals will be exposed to the pesticide product, including, but not
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limited to, exp e through direct application for pest control and con—
s ption of treated feed • The applicant for registration should consult
with the Agency to determine at toxicological data are required. In
some cases, the data resulting fr studies porfozmed on laboratory and
noedaneetic animals can be extrapolated to the dosestic species likely to
be cposed • In these cases, no additional testing will be required.
(b) Testing . Data from any of the studies described in this subdivision
may be required, including, but not Iii { ted to, the following:
(1) Acute oral toxicity;
(2) Acute derma]. toxicity;
(3) Acute inhalation toxicity;
(4) Primary derinal irritation;
(5) Primary eye irritation;
(6) Dermal sensitization;
(7) Subchronic oral dosing;
(8) Cholinesterase inhibition;
(9) Neurotoxiciby; and
(10) Teratogenicity.
(c) Standards • Eath tee t should be pe rfo ed according to the stan-
dards specified by the Agency. The applicant should also refer to standards
specified in the appropriate sections of this subdivision.
(d) Data reporting and evaluation • (1) The general info rma t ion
required by § 80-4 shall be rapo rted for each tee t • In addition, each
test report shall contain all appropriate data required by the “Data
raporting and evaluation” paragraphs of the corresponding sections of this
subdivision.
(2) In addition, the appUcant should subeit any evidence of toxic-
ological effects of the pesticide to d es tic anim la observed during
product performance testing including, in particular, field testing.
§ 85—3 Derma]. Absorption Studies of Pesticides . (Reserved]
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