DRAFT I.R.LG. GUIDELINES
FOR
SELECTED ACUTE TOXICITY TESTS
Testing Standards ft Guidelines Work Group
INTERAGEIMCY REGULATORY LIAISON GROUP
1979
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FOR FURTHER INFORMATION CONTACT:
INDUSTRY ASSISTANCE OFFICE (TS-799)
U. S. ENVIRONMENTAL PROTECTION AGENCY
401 M Street, S.W.
Washington, B.C. 20460
TELEPHONE TOLL-FREE 800-424-9065
OR IN WASHINGTON 554-1404
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PREFACE
This package of "Draft I.R.L.G. Guidelines for Selected
Acute Toxicity Tests" represents the work of the members of the
Testing Standards and Guidelines Work Group of the Interagency
Regulatory Liaison Group (I.R.L.G.). It reflects the views of
staff members of the I.R.L.G. agencies who reviewed earlier drafts
and is being released to obtain public comment before final drafts
are prepared for submission to the I.R.L.G. agencies. The tests in
this package are the first in a series on toxicity testing which
will include other acute as well as chronic effects tests.
On August 21, 1979, the I.R.L.G. published a Federal Re-
gister notice announcing the public availability of these five
draft guidelines and a meeting for public participation in dis-
cussing them. That meeting will be held on October 30 at 9:00 A.M.
in the Auditorium, Main Floor, of the Hubert H. Humphrey Building,
200 Independence Avenue, S.W., Washington, D.C., 20201. Work
Group members will discuss their philosophy, comparisons of these
with other similar guidelines, the relationship of I.R.L.G. guide-
lines to other testing requirements, and future activities of the
Work Group. The public will be invited to participate in this
discussion. In addition, comment by attendees will "be requested on
the scientific issues raised at the beginning of each guideline.
The Work Group has asked that written comments be submitted
by October 19, 1979, to allow some discussion of the'm at the public
meeting. However, the public is encouraged to submit comments after
this suggested deadline as input to the Group's on-geing work. They
should be addressed to Dr. Victor Morgen Roth III, HFF-185, Food
and Drug Administration, Bureau of Foods, Division of Toxicology,
200 "C" Street, S.W., Washington, D.C., 20204. Comments may be
examined in the I.R.L.G. office, Room 509, 1111 18th Street, N.W.,
Washington, D.C., 20207, 9:00 A.M. to 4:00 P.M., Monday through
Friday.
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Testing Standards and Guidelines Work Group
Interagency Regulatory Liaison Group
Chairman: Dr. Victor Morgenroth, III, Food and Drug Administration
Surrogate Liaison: Dr. Allen H. Heim, Food and Drug Administration
Members: Health Effects Group
Dr. James R. Beall
Dr. William D'Aguanno
Dr. Robert Hehir
Dr. Zdenka Horokova
Dr. Patricia Marlow
Dr. Joseph McLaughlin
Dr. Robert E. Osterberg
Dr. Norbert Page
Dr. Orville E. Paynter
Ms. Peggy Perry
Dr. John A. Quest
Mr. Van Seabaugh
Dr. Frode Ulvedal
Occupational Safety § Health Admin.
Food and Drug Administration
Consumer Product Safety Commission
Food Safety § Quality Service
Occupational Safety § Health Admin.
Consumer Product Safety Commission
Food and Drug Administration
Environmental Protection Agency
Department of Commerce
Food and Drug Administration
Food and Drug Administration
Consumer Product Safety Commission
Environmental Protection Agency
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TABLE OF CONTENTS
Preamble 1
Draft I.R.L.G. Guideline for
Acuate Eye Irritation Tests 9
Draft I.R.L.G. Guideline for
Acuate Oral Toxicity Studies in Rodents .... 39
Draft I.R.L.G. Guideline for
Acute Dermal Toxicity Tests 61
Draft I.R.L.G. Guideline for
Acute Inhalation Tests in Rats 87
Draft I.R.L.G. Guideline for
Teratogenicity Testing in Rat, Mouse
and Rabbit 113
ill
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IV
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PREAMBLE
I. Background
Four regulatory agencies, the Consumer Product Safety
Conmission (CPSC), the Environmental Protection Agency (EPA), the
Food and Drug Administration (FDA), and the Occupational Safety
and Health Administration (QSHA), agreed to work together to re-
form the regulatory process and to improve protection of workers,
public health, and the environment (42 FR 54856, 11 October 1977).
They formed the Interagency Regulatory Liaison Group (IRLG) to
implement their agreement. In January 1979, the Food Safety and
Quality Service (FSQS), Department of Agriculture, joined the
IRLG.
These agencies recognized that although they often regulate
the same chemicals, toxicity testing guidelines used by each
agency are not always uniform. Among currently required tests,
differences exist primarily in details of methodology and not in
fundamental toxicological principles. The Testing Standards and
Guidelines Work Group was established for the purpose of develop-
ing guidelines which would resolve existing differences and be
used by all of the IRLG agencies for testing chemicals for health
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or environmental effects or both. The plan ,was to review current
tests and procedures in use or under development and prepare a
single set of toxicity guidelines that could serve the IRLG
agencies. This effort would be coordinated with the development of
other guidelines. On December 17, 1977, the Work Group held a
public neeting to explain its purpose and goals, and to answer
questions about its activities (42 FR 59106, 15 November 1977).
II. Philosophy
The Work Group agreed upon the following tenets:
A. Guidelines must be sufficiently comprehensive to provide
a sound, scientific method for gathering data necessary for char-
acterization of the test substance.
B. Requirements of the guidelines must be feasible.
C. Guidelines must provide adequate guidance to investiga-
tors.
D. Guidelines must be flexible, allowing the investigator
latitude for scientific judgment.
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E. Each guideline should be complete in itself and be able
to stand alone.
F. Guidelines should avoid irrelevant or marginally useful
procedures, while retaining the value of the test. Each recom-
mended procedure should result in data essential for characteriza-
tion of the test substance and useful for regulatory decisions.
G. Costs of conducting the tests must be considered and
kept to a minimum without jeopardizing the validity of the test.
H. Guidelines should be reviewed annually, and opportunity
must be provided for modifications which improve the test and
incorporate advances in the state of the art.
I. Guidelines should be constructed so that they can be
harmonized with those under development nationally and
internationally.
J. Welfare of the test anijnals must be considered.
K. Decisions of the Work Group are made by consensus.
Guidelines recommended by the work group are acceptable to every
member.
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III. Development
IRIJG guidelines are based on those currently used or under
development in the agencies, in international organizations, and
in industry. Early drafts of the IRD3 guidelines were circulated
through each agency for review, and comments from that review are
included in this proposed guideline. In addition, drafts of the
EPA Office of Pesticides Programs (OPP) proposed guidelines (43 FR
37336, 22 August 1978) under the Federal Insecticide, Fungicide
and Rodenticide Act (FIFRA), and public comments to those guide-
lines were used extensively in an effort to assure compatibility
and to benefit from the appreciable ,time and effort the OPP staff
put in to their development. A second major source of information
was Principles and Procedures for Evaluating the Tbxicity of
Household Substances (NAS Pub. No. 1138), prepared for CPSC by the
National Academy of Sciences. Still others heavily relied on were
the Pharmaceutical Manufacturers' Association Guidelines for the
Assessment of Drug and Medical Device Safety in Animals (February
1977)the FDA, Bureau of Foods Direct Additive Cyclic Review Draft
Guidelines; the Appraisal of the Safety of Chemicals in Foods,
Drugs and Cosmetics, Association of Food and Drug Officials of the
U. S; and protocols submitted by several industrial organizations
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in response to 43 FR 1987, 13 January 78. To the extent possible,
IRLG guidelines are being coordinated with development of guide-
lines by the Organization for Economic Cooperation and Development
(OECD). The Work Group acknowledges the contributions from each
of these information sources and takes this opportunity to express
its appreciation to the scientists who developed them and made
then available.
IV. Relationship to Other Guidelines
A. Previously Issued Guidelines and Tests In Progress
To assure that issuance of the IRD3 guidelines will not
cause confusion because other guidelines are being developed to
meet specific agency needs and will not negate tests already in
progress, the IRLG agencies published the following notice (42 FR
1528, 10 January 1978): "To the extent permitted by each agency's
legislation, parts of published standards, regulation, and/or
guidelines may be amended to agree with uniform testing standards
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and guidelines developed by the IRLG. In that event, data result-
ing from use of pre-existing guidelines will be accepted by the
agency requiring them so long as these data are generated from
studies begun before the IRLG guidelines are promulgated and the
data are valid and scientifically sound."
B. Use and Modification of IRLG Guidelines
Use of this guideline will provide test methods for
acquisition of data acceptable to all of the IRLG agencies. Under
most circumstances, the data obtained fron this test should be
sufficient to meet the requirements for a specific aspect of the
toxicological characterization of the test substance. Under cer-
tain circumstances, however, the data nay show a need for further
study; or it may be recognized at the outset this guideline may
have to be modified by an agency. If the modification requires
information in addition to that required by this guideline, the
data will be acceptable to all the IRLG agencies. If the modifi-
cation requires less than this guideline, the data from the test
may not be acceptable to all IRLG agencies. It is important that
modifications be agreed upon between the investigator and the
agency requiring than.
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Each agency will decide how it will use IRLG guidelines.
and has made a commitment to adopt them to the fullest extent
possible consistent with its regulatory responsibilities. For
example, in its proposed guidelines (43 FR 37337, 22 August 1978),
the EPA Office of Pesticide Programs stated that, "At such time as
these conmittees complete their work, EPA will review the IRLG
/
documents and revise its PIFRA guidelines if appropriate."
V. Agency Responsibilities
Each agency has the responsibility to decide:
A. what substance, and what form of the substance, it
requires to be tested;
B. which tests it will require;
C. how it will use the data derived from the tests; and
D. how it will ijnplement use of IRLG guidelines.
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DRAFT I.R.L.G. GUIDELINE FOR
ACUTE EYE IRRITATION TESTS
Testing Standards § Guidelines Work Group
INTERAGENCY REGULATORY LIAISON GROUP
May 30, 1979
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PREFACE
This guideline delineates test procedures to evaluate the
toxicity of liquids, solids, aerosols, and liquids propelled under
pressure, to ocular tissues of laboratory animals. The test
should demonstrate the potential of a substance to produce injury
to the human eye. Evaluation of gases for eye irritation requires
special techniques which are not specified in this guideline.
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{Scientific Issues for Public Ctanment
During the development of this proposed guideline, many scien-
tific issues were discussed by the Work Group. These issues were
raised by members of the Work Group, by public cornnents to the EPA
proposed pesticide guidelines, and by the conments of interagency
reviewers. Consideration of these comments, discussed below, is
reflected in this proposed guideline; and the public is invited to
conment further on these issues or any other aspect of this proposed
guideline.
A. Either males or females may be used for this test. Although
a review of available data indicates that eye irritation is not a sex
dependent response, sane carmentors have suggested that equal numbers
of both sexes should be used. Information about which sex, if
either, is more appropriate for eye irritation studies and data show-
ing sex differences, or lack of differences, in response to eye irri-
tants would be helpful.
B. The Work Group chose the albino rabbit as the preferred
animal for this test, although the rabbit's lesser predictive
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ability for human ocular irritancy with respect to other species,
such as monkeys is recognized. Hie Work Group solicits comments
regarding the use of ocular irritancy data obtained from other
species and whether they should take precedence over rabbit data.
Discussion of the use of other animals should include difficulty of
obtaining them, cost, ease of handling, and structural differences of
ocular tissues.
C. The Work Group realizes that the classical method of
instillation of the test substance in eye irritation studies is into
the cul-de-sac. Sane canmentors raised the issue of approximating
human responses and decreasing exaggerated rabbit responses by admin-
istration of the test substance directly onto the cornea, rather than
into the conjunctival sac. The Work Group would like information
concerning the predictive nature of each procedure in approximating
the human response and which procedure produces responses that take
into account the more susceptible members of the population in terms
of potential irritancy.
D. The Work Group reviewed comments on the OPP guidelines re-
garding the amount of test substance that would be expected to con-
tact human eyes in accidental situations and its relevance to the
volume applied to rabbit eyes in the eye irritation test. The Work
Group realizes that the use of several volumes of test material (0.01
ml to 0.1 ml) applied to rabits better delineates the dose-response
characteristics of an irritant. The purpose of this guideline, how-
ever, is to detect irritance; and therefore the use of a single,
large volume
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has been recommended. The 0.1 ml volume closely approximates 2
drops, a realistic volume of exposure that a human eye might
receive.
E. "Hie Work Group recognizes the possibility of a traumatic
response from instillation of a test substance into the eyes from an
aerosol spray. Coranents are solicited on use of an alternate method
whereby the aerosolized material is collected in a chilled container
and tested identically as with the other liquids not propelled under
pressure.
F. Several comments to the OPP proposed guidelines were
received concerning the examination of eyes 24 hours prior to
instillation of the test material. Since eye damage could occur
within the 24 hour period making the animal unacceptable for testing,
a shorter time interval, such as 1 or 4 hours, was suggested in order
to minimize this potential. The Work Group decided that the phrase
"within 24 hours" would accomodate this concern, while allowing the
investigators to use their own judgement.
G. Another topic at issue is the requirement to hold rabbits
beyond 72 hours even though no responses have occurred or those that
have, have disappeared. The Work Group is unaware of chemicals that
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that produce delayed type of ocular irritation following one expos-
ure. Comments on the need for observations beyond 72 hours are
solicited.
G. Properly used, fluorescein or other stains highlight
changes in tissue in eye irritation studies. Improperly admin-
istered, such stains can contaminate the eyes with bacteria or irri-
tating materials. In this guideline, the use of such stains is
proposed to be optional in the examination of the eye.
H. Anesthetics may obscure a pain response to the irritant,
but rarely interfere with the response of an eye to an irritant;
therefore, the Work Group proposes that for humane reasons anes-
thetics should be used when the substance being tested is likely to
cause extreme pain. Information about the effects, including effects
on healing time, of one instillation of a local anesthetic in
altering responses of the tissues to eye irritants is solicited.
I. Terata have been induced in offspring following the instil-
lation of glucocorticoids to the eyes of pregnant rabbits. This and
other evidence indicate that substances can be absorbed following
ocular instillation; however, systemic toxicity is usually not
evaluated in acute eye irritation tests. The Work Group invites
views on whether to require the evaluation of systemic effects in the
acute eye irritation test.
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evidence indicate that substances can be absorbed following ocular
instillation; however, systemic toxicity is usualy not evaluated in a
cute eye irritation tests.
J. The issue of the most appropriate scoring system was
discussed. The Work Group felt that the widely used scoring system
utilized in this guideline permits a relatively realistic classifica-
tion of degrees of hazard, is less subject to the distortion that
occurs using a weighted system, and is more sensitive to subtle
ocular effects.
K. It was suggested that substances be tested in diluted form
and also that substances be washed from the eyes soon after admin-
istration in order to evaluate the potential of a substance to cause
irritation under conditions of normal use. Data obtained from eyes
washed following instillation of the substance were considered to be
indicative more of the value of possible first aid treatment than of
the potential of the substance to cause eye irritation. The Work
Group also suggests that additional studies may be appropriate for
shampoos or other substances which, in normal use, might enter the
eye in diluted form or might be washed out immediately. The Work
Group thinks that such studies could be useful, but should be done in
addition to an initial eye irritation test of the neat substance.
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TABLE OF CONTENTS
I. General Considerations
A. Good Laboratory Practices
B. Personnel
C. Test Substance
D. Animals
E. Dead Animals/ Necropsy, and Histopathology
F. Equipment
G. Documentation
II. Specific Considerations
A. Test Preparation
B. Test Procedure
III. Data Reporting
A. Identification
B. Body of Report
1. Summary and Conclusions
2. Materials
3. Methods
4. Results
5. References
IV. Suggested Reading
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I. General Considerations
A. Good Laboratory Practices
Basic standards presented here relating to good labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive. This guide1-
line does not set forth the managerial aspects of science or good
laboratory practices. Studies should be conducted according to
"Nonclinical Laboratory Studies, Good Laboratory Practice
Regulations," (43 FR 59986, 22 December 1978).
B. Personnel
All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures. The agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing. To the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
tency of evaluation. When a histopathological examination is
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done, similar considerations should apply.
C. Test Substance (materials or mixtures of substances or
materials)
1. As far as is practical, composition of the test
substance must be known, including the name and quantities of
known contaminants and impurities. Unknown materials, if any,
must be quantified to account for 100% of the test sample. The
specific substance to be tested will be determined in consultation
with each agency.
2. The lot of the substance tested should be the same
throughout the study. The test sample should be stored under
conditions that maintain its stability, strength, quality, and
purity from the date of its production until the tests are
complete.
3. Safe handling and disposition of the test substance
is essential.
D. Animals
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1. Animals used for testing should not have been
subjected to any previous experimental procedures.
2. The test animal shall be characterized as to
species, strain, sex, weight and/or age. Each animal must be
assigned an appropriate identification nunber.
3. Recommendations contained in DHEW pub. no. (NIH)
74-23, entitled "Guide for the Care and Use of Laboratory
Animals," should be followed for the care, maintenance, and
housing of animals.
4. Animals may not be group-caged for this test.
5. Healthy animals must be used. Animals must be
assigned to groups in such a manner as to minimize bias and assure
comparability of pertinent variables.
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6. Each animal must be observed as necessary to insure
that animals are not lost due to autolysis of tissues, misplace-
ment, or similar management problems.
E. Dead Animals, Necropsy, and Histopathology
When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately. Necropsy must be performed with-
in 16 hours of death. When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopathological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.
F. Equipment
All equipment used in conducting the test, including
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equipment used to prepare and administer the test substance and
equipment used to maintain environmental conditions, must be of
appropriate design and adequate capacity. Equipment should be
inspected, cleaned, and maintained regularly. The equipment must
be properly calibrated at the time of its use.
G. Documentation
Color photograpic documentation to verify gross and
microscopic findings or to clarify conflicting data is a desirable
aspect of ocular toxicity studies. If photographs are taken, the
equipment and film must be of sufficient quality to permit con-
trolled, close-up color photography of the eye to yield clear,
sharp-focus images that literally fill the camera field.
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II. Specific Considerations
A. Test Preparation
1. Testing shall be performed on either male or female
albino rabbits weighing between 2.0 and 3.0 kilograms. Other
species may also be tested for oomparative purposes.
2. The nunber of animals to be tested for each test
substance must be adequate for analysis. At least 6 rabbits must
survive the test for each test substance. If additional testing
is necessary for estimating dose response or for further evalua-
tion, more animals will be required.
3. Animal facilities should be so designed and main-
tained as to exclude sawdust, wood chips, or other extraneous
materials that might produce eye irritation. In addition, animals
under test should not be exposed for long periods to intense and
direct light, as it may damage the retina.
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B. Test Procedure
1. Both eyes of each animal in the test groups must be
examined (by use of optical instruments, fluorescein, ultraviolet
light, or other appropriate means) within 24 hours before subs-
tance administration. Animals with eye defects or irritation must
be excluded.
2. For most purposes, anesthetics should not be used;
however, if the test substance is likely to cause extreme pain,
local anesthetics may be used for humane reasons. In such cases,
anesthetics should be used only once, just prior to instillation
of the test substance; the eye used as the control in each rabbit
should also be anesthetized. Proparacaine 0.5% and butacaine
sulfate 2% are acceptable anesthetics.
For substance administration, the animal is held
firmly but gently until it appears to be quiet. The test sub-
stance is placed in one eye of each animal by gently pulling the
lower lid away from the eyeball (conjunctival cul-de-sac) to form
a cup into which the test substance is dropped. The lids are then
gently held together for one second and the animal is released.
The other eye, remaining untreated, serves as a control. Vehicle
controls are not included. If a vehicle is suspected of causing
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irritation, additional studies should be conducted, using the
vehicle as the test substance.
3. For testing liquids, 0.1 milliliter is used. For
solids or pastes, 100 miligrams of the test substance is used.
For participate substances (flake, granule, powder, or other
particulate form), the amount used must have a volume of 0.1
milliliter weighing not more than 100 mg. The measure should be
taken after gently compacting the particulates by tapping the
measuring container in a way that will not alter their individual
form. The weight of the 0.1 milliliter test dose must be
recorded.
4. For aerosol products, the substance should be
administered as a single, short burst of about one second at a
distance of about 4 inches directly in front of the eye (held
open), provided that the distance insures that the velocity of the
ejected material does not traumatize the eye. The dose should be
approximated by weighing the aerosol can before and after each
treatment. For other liquids propelled under pressure, such as
substances delivered by pump sprays, an aliquot of 0.1 ml should
be collected and instilled in the eye as for liquids.
The eyes are not washed following instillation of
the test substance, except as noted for fluorescein staining.
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After the 24-hour examination, the eyes may be washed, if desired.
Tap water or isotonic solution of sodium chloride (U.S.P.) should
be used for all washings.
For some substances (such as shampoos) shown to be
irritating by this test, additional tests using rabbits with eyes
washed soon after instillation of the substance may be needed. In
these cases, it is recommended that 6 rabbits be used. Pour
seconds after instillation of the test substance, the eyes of 3
rabbits are washed, and at 30 seconds after instillation, the eyes
of the other 3 are washed. For both groups, the eyes are washed
for five minutes using a volume and velocity of flow that are not
traumatizing.
C. Observations
1. The eyes should be examined at 1, 24, 48, and 72
hours, and 7 days after treatment. In addition to the required
observations of the cornea, iris, conjunctivae, serious lesions
such as pannus, phlyctena, and rupture of the globe should be
reported. The grades of ocular reaction (Table I) must be
recorded at each examination. If the cornea, iris, or conjunctivae
has not healed completely by the seventh day, the unhealed animals
should be retained and re-examined on the 14th day, and again at
the 21st day if injury persists. Evaluation of reactions can be
facilitated by use of a binocular loupe, hand slit-lamp, or other
expert means.
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2. After the recording of observations at 24 hours,
the eyes of any or all rabbits may be further examined after
applying flourescein stain. For this optional examination, one
drop of flourescein sodium ophthalmic solution (U.S.P) is dropped
directly on the cornea. After flushing out the excess flourescein
with tap water or isotonic solution of sodium chloride (U.S.P.),
injured areas of the cornea appear yellow. These changes are best
seen under ultraviolet illumination in a darkened room.
3. A record of the discharge from treated eyes is not
required; however, any exudate above normal can be recorded as
additional information.
4. An animal has exhibited a positive reaction if the
test substance has produced at any observation one or more of the
following signs:
(a) ulceration of the cornea (other than a fine
stippling)
(b) opacity of the cornea (other than a slight
dulling of the normal luster),
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(c) inflammation of the iris (other than a slight
deepening of the rugae or a light hyperemia of the circumcorneal
blood vessels), or
(d) an obvious swelling in the conjunctivae
(excluding the cornea and iris) with partial eversion of the
eyelids and a diffuse crimson color with individual vessels not
easily discernible.
5. Table I - Grades for Ocular Lesions
The grading of ocular responses is subject to vari-
able interpretations. To promote standardization and to assist
in interpreting the observations in accordance with this guide-
line, a training film, entitled "Laboratory Procedures for Testing
Eye Irritation," (Digest No. 10237) and an "Illustrated Guide for
Grading Eye Irritations" (Digest No. 10239) have been prepared. A
limited number of copies of the guide are available from the
Consumer Product Safety Commission Directorate for Engineering and
Science, Washington, D. C. 20207. The film is available on loan
from Modern Talking Pictures, Inc., 2000 "L" Street, N.W.,
Washington, D. C. 20036. Copies of the film (Identification No.
CPSC M51361) can be purchased from Movielabs, Inc., Movielabs
Building, 619 West 54th Street, New York City 10019.
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TABLE I
Grades for Ocular 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 areas/ details of
iris slightly obscured 2
Nacreous areas, no details of iris visible, size of pupil
barely discernible , 3
Opaque cornea, iris not discernible through the opacity 4
IRIS
Normal n
Markedly deepened rugae, congestion, swelling, moderate
circumcorneal hyperemia, or injection, any of these
or combination any thereof/ iris still reacting to
light (sluggish reaction is positive) , 1
No reaction to light, hemorrhage, gross destruction (any
or all of these) ">.
CONJUNCTTVAF;
Redness (refers to palpebral ad bulbar conjunctivae excluding
cornea and iris)
Blood vessels normal , , , , , ,,,..,.... 0
Some blood vessels definitely hyperemic (injected) I
Diffuse, crimson color, individual vessels not easily discernible. ..2
Diffuse beefy read 3
Chemosis: lids and/or nictitating membranes
No swelling 0
Any swelling above normal (includes nictitating membranes) .1
Obvious swelling with partial eversion of lids 7.
Swelling with lids about half closed ,, .3
Swelling with lids more than half closed 4
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D. Classification of Test Substances
Classification*
fteaction
Non-irritant
No positive reactions
(opacity, iritis or conjunctiv-
itis on more than 1 out of 6
test animals at 1-3 days and
all eyes normal at 7th day.
Irritant
Opacity grades 1.0 to 2.0 at
any observation up to 7 days.
All corneas cleared at 14
days.*
Iritis 1.0 at 1-7 days, but all
iritis cleared by 14th day.*
Conjunctivitis:
Redness grade 2.0 at 1-7 days
Chemosis grades greater than
2.0 at 1-7 days.
* If not cleared at 14 days, substance is considered a severe
irritant.
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Severe
Irritant or
Corrosive**
Opacity greater than 2.0.
Injury persists 14-21 days.
Corneal perforation or
necrosis at any observation
period.
Pannus or phlyctenular
reactions.
Iritis grade greater than 1.0
at 1-7 days, all eyes not
clear at 14 or 21 days.
Conjunctivitis:
Badness grades greater than
2.0 at 1-7 days.
Chemosis grades greater than
2.0 at 1-7 days.
** Opacity grades 2 to 4 and/or perforation of the cornea are
considered to be corrosive effects when opacities persist to 21 days.
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III. Data Reporting
A. Identification
Each test report must be signed by the person
responsible for the test and identify:
1. The laboratory where the test was performed by
name and address;
2. The inclusive dates of the test; and
3. Each person primarily responsible for separate
ccraponents of the test and the component for which the person is
reponsible including (a) the conduct of the test, (b) analysis of
the data, (c) the writing of the report, and (3) any written or
other matter contained in the report.
B. Body of Report
The test report must include all information necessary
to provide a complete and accurate description and evaluation of
31
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E. Conclusions
1. The test shall be considered positive if four or more of
the animals in either of the test groups (rabbits with eyes washed, or
with eyes unwashed) exhibit a positive reaction. If only one animal
exhibits a positive reaction, the test shall be regarded as negative.
If two or three animals exhibit a positive reaction, the toxioologist
in charge of the test may designate the substance to be an irritant.
If he/she does not, the test shall be repeated using a different group
of six animals. The second test shall be considered positive if three
or more of the animals exhibit a positive reaction.
2. If only one or two animals in the second test exhibit a
positive reaction, the test should be repeated with a different group
of six animals. When a third test is needed, the substance will be
regarded as an irritant if any animal exhibits a positive response.
32
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the test procedures and results. Each report must include the
following sections:
1. Summary and Conclusions. This section of the test
report should contain a tabular summary of the data, an analysis
of the data, and a statement of the conclusions drawn from the
analysis. The summary must highlight all positive data and
observations and any other indications of toxic effects.
2. Materials. This section of the test report shall
include, but not be limited to/ the following information:
(a) Identification of the test substance,
including:
i. chemical name, molecular structure, and a
qualitative and quantitative determination of its chemical
composition, including names and quantities of known contaminants
and impurities, so far as is practical; the determinations shall
also include a listing of materials as unknowns, if any, so that
100% of the test sample is accounted for;
33
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ii. manufacturer and lot nunber of the substance
tested, and such information as physical state, pH, stability, and
purity; and
iii. exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.
(b) Animal data, including:
i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;
ii. source of supply of the animals;
iii. description of any pre-test conditioning,
including diet;
iv. description of the method used in
randomization of animals to test groups; and
v. nunbers of animals of each sex in each test
group.
34
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(c) Data on facilities should include description
of the caging conditions including bedding material, ambient
temperature, and humidity.
3. Methods
(a) Deviation from guidelines - This section
shall indicate all ways in which the test procedure deviates from
this guideline and shall state the rationale for such deviation.
(b) Specification of test methods - This section
shall include a full description of the experimental design and
procedure, the length of the study, and the dates on which the
study began and ended.
(c) Statistical analysis - All statistical methods
used should be fully described or identified by reference.
(d) Data on dosage administration, including:
i. all dose levels administered;
35
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ii. method and frequency of administration; and
iii. total volume of substance (i.e., test
substance plus vehicle) contained in individual dosages.
(e) Data on observation methods, including:
i. duration; and
ii. method and frequency of observation of the
animals.
4. Results
The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results, including summaries and tables that show
the relationship of effects to time of dosing, sex, etc.
36
-------
5. References
This section of the test report shall include the
following information:
(a) Availability of original data, specimens and
samples of the test substance. The location of all original data,
specimens, and samples of the test substances vrfiich are retained
in accordance with the testing requirement.
(b) Literature or references, including, where
appropriate, those references for (1) test procedures, (2)
statistical and other methods used to analyze the data, (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
IV. Suggested Reading
1. Draize, J. H., 1959. Dermal Toxicity. In; Association
of Pood and Drug Officials of the U. S. Austin, Texas. Appraisal
of the Safety of Chemicals in Foods, Drugs, and Cosmetics, pp.
46-59.
37
-------
2. Green, W. R., J. B. Sullivan, R. M. Hehir, and L. F.
Scharpf. A systematic comparison of chenically-induced eye injury
in the albino rabbit and rhesus monkey In; The Soap and Deter-
gent Association. Submission to the National Academy of Sciences
by the Soap and Detergent Association on toxicity test procedures
with Appendices A-P. Appendix C.
3. National Academy of Sciences - National Research
Council, 1977. In; Principles and Procedures for Evaluating the
Toxicity of Household Substances, Report No. 1138, prepared for
the Consumer Product Safety Commission. Eye Irritation, pp.
62-91.
4. Prince, J. H., 1964. The Rabbit in Eye Research.
Springfield, 111. Charles C. Thomas.
38
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DRAFT I.R.L.G. GUIDELINE FOR
ACUTE ORAL TOXICITY STUDIES IN RODENTS
Testing Standards § Guidelines Work Group
INTERAGENCY REGULATORY LIAISON GROUP
May 30, 1979
39-
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PREFACE
The LDjQ value is the nost frequently determined index of
toxicity and is required by some Federal legislation.
Although several accepted methods for determining the LD5Q
values have been developed, many important determinants of tox-
icity are not represented either by these values or slopes of
dose-response curves for lethality. These determinants are inte-
gral to an evaluation of acute toxicity and should be observed
during the course of an acute toxicity study. Site and mechanisn
of action, early or delayed death, and recovery rate may be better
indices of toxicity and hazard than LE^g values per se. Mor-
bidity and/or pathogenesis may have more toxicological signifi-
cance than mortality.
This guideline is designed for use in acute ingestion tests using
rodents, but is adaptable by example to other species.
40
-------
Scientific Issues for Public Conttient
During the development of this proposed guideline, many scientific
issues were discussed by the Work Group. These issues were raised by
members of the Work Group, by the public comments to the EPA proposed
pesticide guidelines and by the comments, of interagency reviewers.
This proposed guideline reflects the Work Group's consideration of
these conments. Consideration of these comments, discussed below, is
reflected in this proposed guideline; and the public is invited to
comment further on these issues or any other aspect of this proposed
guideline.
A. Several commentors suggested at least 5 to 6 dose levels in
order to achieve reliable mortality and obtain appropriate confidence
limits of the LD50 value. The Work Group feels that four dose
levels, when properly spaced, should provide sufficient data for
estimations.
B. Many commentors felt that restricting the 95% confidence
limits of the LD$Q to plus or minus 20% or less may be too
restrictive. Comments are solicited concerning the limitation of the
confidence limits to 20%.
41
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C. The Work Group recognizes the difficulty of selecting dose
levels which will produce mortality rates between 10 and 90% and
requests comments concerning methods of dose selection for acute oral
toxicity studies.
D. The Work Group thinks that fasting of animals is necessary to
obtain more uniform absorption of the test substance in the acute oral
study, but the period of fasting should not be so long as to induce
significant stress (metabolic or otherwise) in the test animals.
Comments are requested concerning the effect of fasting on acute oral
toxicity and appropriate fasting periods for various species.
E. Many comments were received concerning the frequency of
clinical (visual) observations of the test animals. The Work Group
thinks that observation at least twice a day following the day of
substance administration is necessary in order to determine the acute
toxicity profile of the test substance. Comments on the frequency of
clinical observations are requested.
42
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TABLE OF CONTENTS
I. General Considerations
A. Good Laboratory Practices
B. Personnel
C. Test Substance
D. Animals
E. Dead Anijnals, Necropsy, and Histopathology
F. Equipment
II. Specific Considerations
A. Test Preparation
B. Test Procedure
III. Data Reporting
A. Identification
B. Body of Report
1. Summary and Conclusions
2. Materials
3. Methods
4. Results
5. References
IV. Suggested Reading
43
-------
I. General Considerations
A. Good Laboratory Practices
Basic standards presented here relating to good labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive. This guide-
line does not set forth the managerial aspects of science or good
laboratory practices. Studies should be conducted according to
"Nonclinical Laboratory Studies/ Good Laboratory Practice
Regulations," (43 FR 59986, 22 December 1978).
B. Personnel
All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures. The agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing. To the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
tency of evaluation. When a histopathological examination is
44
-------
done, similar considerations should apply.
C. Test Substance (materials or mixtures of substances or
materials)
1. As far as is practical, composition of the test
substance must be known, including the name and quantities of
known contaminants and impurities. Unknown materials, if any,
must be quantified to account for 100% of the test sample. The
specific substance to be tested will be determined in consultation
with each agency.
2. The lot of the substance tested should be the same
throughout the study. The test sample should be stored under
conditions that maintain its stability, strength, quality, and
purity from the date of its production until the tests are
complete.
3. Safe handling and disposition of the test substance
is essential.
D. Animals
45
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1. Animals used for testing should not have been
subjected to any previous experimental procedures.
2. The test animal shall be characterized as to
species, strain, sex, weight and/or age. Each animal must be
assigned an appropriate identification number.
3. Recommendations contained in DHEW pub. no. (NIH)
74-23, entitled "Guide for the Care and Use of Laboratory
Animals," should be followed for the care, maintenance, and
housing of animals.
4. Animals may be group-caged for this test unless the
pharmacological action of the test substance dictates otherwise.
However, the number of animals per cage should not prevent
continued and clear observation of each animal. When signs of
morbidity or excitability are observed in group-caged animals
during the test, such animals should be moved to separate cages.
5. Healthy animals must be used. Animals must be
assigned to groups in such a manner as to minimize bias and assure
comparability of pertinent variables.
46
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6. Each animal must be observed as necessary to insure
that animals are not lost due to cannibalism, autolysis of
tissues, misplacement, or similar management problems.
7. When control animals are used, they must be housed,
fed, and handled exactly like the test animals; and they must be
caged to minimize airborne or other contamination by the test
substance.
E. Dead Animals, Necropsy, and Histopathology
When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately. Necropsy must be performed with-
in 16 hours of death. When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopathological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.
F. Equipment
All equipment used in conducting the test, including
47
-------
equipment used to prepare and administer the test substance and
equipment used to maintain environmental conditions, must be of
appropriate design and adequate capacity. Equipment should be
inspected, cleaned, and maintained regularly. The equipment must
be properly calibrated at the time of its use.
48
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II. Specific Considerations
A. Test Preparation
1. Animals: Laboratory strains of rats (125-250 g each)
and/or mice (20-30 g each) should be used. When attempting to estimate
hazards to young humans, additional studies designed to consider the
developmental stage of the test animal in relation to anticipated human
exposure should be performed.
2. Number and sex: At least 10 animals, 5 per sex, random-
ly assigned should be used at each dose level. The females should be
nonpregnant.
3. Controls: Untreated controls are generally not re-
quired, since dose reponse during an LDjg may serve as an internal
control. A negative or vehicle control group is not required; however,
if a vehicle or solvent of uncharacterized toxic potential is used, an
acute oral toxicity test should be done on the solvent.
4. Dose levels: At least four dose levels should be used,
spaced appropriately to produce test groups ideally with mortality
rates between 10% and 90% to permit the calculation of the U)$Q
value for males and females with a 95% confidence limit. Where
possible, the 95% confidence limit should not exceed approximately plus
or minus 20% of the ID value.
49
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5. Fasting: Animals should be fasted prior to admin-
istration of test substance. Pood should be withheld from rats
overnight; from mice for 6 to 8 hours.
B. Test Procedure
1. Dosage: For an acute study, one or more doses of
the test substance may be administered within a 24-hour period.
Ideally the substance should be administered in a single dose.
The determination of Ll^g values of insoluble solids can be
difficult because of the nature of the suspension that may have to be
administered. Such limitations may be circumvented, when necessary, by
the administration of the test substance in divided doses over a period
of several hours. An adequate estimate of acute hazard is obtained for
most purposes if data based on this test is submitted showing that the
value of 5gA9-
2. Route of administration: The dose should be admin-
istered by gavage, not in the food. The dose is administered via
soft rubber or polyethylene tubing or a large ball-tip needle.
The maximum volume of liquid that can be given depends on the
rodent's size and should not exceed 2 ml/lOOg body weight. When
50
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possible/ variability in test volume should be minimized, with
concentrations being adjusted accordingly.
3. Observation period: The observation period should be at
least 14 days. Although a 14-day observation period is sufficient for
most compounds, animals demonstrating visible signs of toxicity after
14 days could be held longer.
4. Recording of clinical observations: Observations should
be recorded systematically as they are made. The animals should be
observed frequently during the first day and twice a day thereafter at
least 4 hours apart (once each morning and late afternoon). Individual
records should be maintained for each animal. Visual observations
should include, but not be limited to, changes in skin and fur, eyes,
mucous membranes and respiratory, cardiovascular, autonomic and central
nervous sytems, and somato-motor activities. Particular attention
should be directed to observations for the presence of tremors, convul-
sions, salivation, hyperactivity, diarrhea, lethargy, sleep, coma,
blanching, cyanosis, and vasodilation. The time at which signs of
toxicity appear and the time of death must be recorded.
51
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5. Weight change: Individual weights of animals must
be determined on the day the test substance is administered,
weekly thereafter, and prior to sacrifice.
6. Necropsy: A complete gross necropsy should be per-
formed on all animals that die during the course of the test and all
remaining animals at termination of the test. Gross pathological
changes of the intestinal tract and the major organs such as liver,
kidney, heart, brain, and spleen should be noted. Liver, kidney, and
organs showing evidence of gross pathology of all animals surviving 12
or more hours should be preserved for possible future microscopic
examination.
52
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III. Data Reporting
A. Identification
Each test report must be signed by the person
responsible for the test and identify:
1. The laboratory where the test was performed by
name and address;
2. The inclusive dates of the test; and
3. Each person primarily responsible for separate
components of the test and the component for which the person is
reponsible including (a) the conduct of the test, (b) analysis of
the data/ (c) the writing of the report, and (3) any written or
other matter contained in the report.
B. Body of Report
The test report must include all information necessary
to provide a complete and accurate description and evaluation of
53
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the test procedures and results. Each report must include the
following sections:
1. Summary and Conclusions. This section of the test
report should contain a tabular summary of the data, an analysis
of the data, and a statement of the conclusions drawn from the
analysis. "Hie summary must highlight all positive data or
observations and any deviations from control data which may be
indicative of toxic effects.
2. Materials. This section of the test report shall
include, but not be limited to, the following information:
(a) Identification of the test substance,
including:
i. chemical name, molecular structure, and a
qualitative and quantitative determination of its chemical
composition, including names and quantities of known contaminants
and impurities, so far as is practical; the determinations shall
also include a listing of materials as unknowns, if any, so that
100% of the test sample is acccounted for:
54
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ii. manufacturer and lot number of the substance
tested, and such information as physical state, pH, stability, and
purity; and
iii. exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.
(b) Animal data, including:
i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;
ii. source of supply of the animals;
iii. description of any pre-test conditioning,
including diet;
iv. description of the method used in
randomization of animals to test or control groups; and
v. numbers of animals of each sex in each test
and control group.
55
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(c) Data on facilities should include description
of the caging conditions including number of animals per cage,
bedding material, ambient temperature, and humidity.
3. Methods
(a) Deviation from guidelines - This section
shall indicate all ways in which the test procedure deviates from
these guidelines and shall state the rationale for such
deviation.
(b) Specification of test methods - This section
shall include a full description of the experimental design and
procedure, the length of the study, and the dates on which the
study began and ended.
(c) Statistical analysis - All statistical methods
used should be fully described or identified by reference.
(d) Data on dosage administration, including:
i. all dose levels administered, expressed as
mg/kg of body weight;
56
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ii. method and frequency of administration; and
iii. total volume of substance (i.e., test
substance plus vehicle) contained in individual dosages.
(e) Data on obsevation methods, including:
i. duration; and
ii. method and frequency of observation of the
animals.
4. Results
The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.
(a) Tabulation of the response data (i.e., nunber
of animals dying; number of animals showing signs of toxicity;
nunber of animals exposed) at each exposure level by sex, and time
of death after dosing;
57
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(b) 11)50 values for each test substance
calculated at the end of the observation period, with method of
caluclation specified;
(c) 95% confidence interval for the
values;
(d) Slope of the dose-mortality curve for each
substance tested; and
(e) Findings from all clinical observations,
necropsy, and histopathological examinations (when made).
5 . References
This section of the test report shall include the
following information:
(a) Availability of original data, specimens and
samples of the test substance. The location of all original data,
specimens, and samples of the test substances which are retained
in accordance with the testing requirement.
58
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(b) Literature or references, including, where
appropriate, those references for (1) test procedures, (2)
statistical and other methods used to analyze the data, (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
59
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IV. Suggested Reading
1. Balazs, T. 1970. Measurement of Acute Toxicity. In;
Methods in Toxicology. G. E. Paget, ed. F. A. Davis Co.,
Philadelphia, Pa.
2. Hagan, E. C. 1959. Acute Toxicity. Jrp Appraisal of the
Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food
and Drug Officials of the United States.
3. Bliss, C. I. 1938. The determination of the dosage mortal-
ity curve from small numbers. Quarterly Journal Pharm. Pharmacol.
11:192-216.
4. Litchfield, J. T., Jr. and F. Wilcoxon. 1949. A simplified
method of evaluating dose-effect experiments. J. Pharmacol. Exp.
Therap. 96:99-115.
5. Thompson, W. R. 1947., Using of moving averages and inter-
polation to estimate median effective dose. Bacteriological Rav.
11:115-145.
60
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DRAFT I.R.L.G. GUIDELINE FOR
ACUTE DERMAL TOXICITY TESTS
Testing Standards § Guidelines Work Group
INTERAGENCY REGULATORY LIAISON GROUP
May 15, 1979
61
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PREFACE
A test for acute dermal toxicity should evaluate the potential for
systemic and local toxic effects of chemicals expected to come in
contact with the skin. The acute dermal test refers to one period
of topical application of up to 24 hours (the exposure period) and
an observation period of at least 14 days.
62
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Scientific Issues for Public Gommsnt
During the development of this proposed guideline, many scientific
issues ware discussed by the work group. These issues were raised by
members of the Work Group, by the public comments to the EPA proposed
pesticide guidelines and by the comments of interagency reviewers.
Consideration of these comments/ discussed below, is reflected in this
proposed guideline; and the public is invited to comment further on
these issues or any other aspect of this proposed guideline.
A. Several commentors stated that dermal toxicity studies should
be conducted on the product as it will be encountered in actual use.
Specifically, granular and pelleted formulations will not come into
contact with the skin as a paste as would occur using the IRD3
guidelines.
The Work Group requests comment on the form in which the test
substance should be applied to the skin.
B. Because of the various methods used to remove fur from the
dermal test areas or the times at which test areas are prepared prior
to application of the test substance, there are potential problems in
the reproducibility of the test responses.
Information regarding the most appropriate method of test site
preparation is requested.
63
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C. Although the use of abraded skin may be apropriate for testing
drugs and cosmetics intended for use on damaged or diseased skin,
abraision techniques have not been standardized; and responses obtained
from substances applied to abraded sites may be variable.
Information is requested concerning the validity of performing
acute dermal toxicity tests using abraded skin and the use of abraded
skin in trial test mortality determinations with 2 gAg or 2 ltd/kg
application of the test substance.
D. Several oommentors suggested that dermal exposure should not be
limited to 24 hours, but should be varied to simulate actual use condi-
tions.
The Work Group requests comments on this issue.
E. Another issue is the frequency of clinical observation of the
test animals. The Work Group feels that at least twice a day observa-
tions is necessary in order to determine the time of onset, duration
any acute effects.
Comments on this topic are invited.
64
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TABLE OF CONTENTS
I. General Considerations
A. Good Laboratory Practices
B. Personnel
C. Test Substance
D. Animals
E. Dead Animals, Necropsy, and Histopathology
F. Equipment
II. Specific Considerations
A. Test Preparation
B. Test Procedure
III. Data Reporting
A. Identification
B. Body of Report
65
-------
1. Summary and Conclusions
2. Materials
3. Methods
4. Results
5. References
IV. Suggested Reading
66
-------
I. General Considerations
A. Good Laboratory Practices
Basic standards presented here relating to good labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive. This guide-
line does not set forth the managerial aspects of science or good
laboratory practices. Studies should be conducted according to
"Nonclinical Laboratory Studies, Good Laboratory Practice
Regulations," (43 PR 59986, 22 December 1978).
B. Personnel
All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures. The agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing. Tto the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
tency of evaluation. When a histppathological examination is
67
-------
done, similar considerations should apply.
C. Test Substance (materials or mixtures of substances or
materials)
1. As far as is practical, composition of the test
substance must be known, including the name and quantities of
known contaminants and impurities. Unknown materials, if any,
must be quantified to account for 100% of the test sample. The
specific substance to be tested will be determined in consultation
with each agency*
2. The lot of the substance tested should be the same
throughout the study. The test sample should be stored under
conditions that maintain its stability, strength, quality, and
purity from the date of its production until the tests are
complete.
3. Safe handling and disposition of the test substance
is essential.
D. Animals
68
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1. Animals used for testing should not have been sub-
jected to any previous experimental procedures.
2. The test animal shall be characterized as to
species/ strain, sexf weight and/or age. Each animal must be
assigned an appropriate identification number.
3. Reccmnendations contained in DHEW pub. no. (NIH)
74-23, entitled "Guide for the Care and Use of Laboratory
Animals," should be followed for the care, maintenance, and
housing of animals.
4. Because it is necessary to prevent oral ingestion
of the test substance, animals may not be group-caged for this
test.
5. Healthy animals must be used. Animals must be
assigned to groups in such a manner as to minimize bias and assure
comparability of pertinent variables.
69
-------
6. Each animal must be observed as necessary to insure
that animals are not lost due to cannibalism, autolysis of
tissues, misplacement, or similar management problems.
7. When control animals are used, they must be housed,
fed, and handled exactly like the test animals; and they must be
caged to minimize airborne or other contamination by the test
substance.
E. Dead Animals, Necropsy, and Histopathology
When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately. Necropsy must be performed with-
in 16 hours of death. When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopathological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.
F. Equipment
All equipment used in conducting the test, including
70
-------
equipment used to prepare and administer the test substance and
equipment used to maintain environmental conditions, must be of
appropriate design and adequate capacity. Equipment should be
inspected, cleaned, and maintained regularly. The equipment must
be properly calibrated at the time of its use.
71
-------
II. Specific Considerations
A. Test Preparation
1. Animals: The young, adult, albino rabbit weighing
2.0 to 3.0 kg is the preferred species because of its size, ease
of handling and restraint, and skin permeability. Selection of
other species may be acceptable but must be justified.
2. Number and sex: Bgual numbers of animals of each
sex with intact skin are required for each dose level. The number
of animals per dose depends on the level of statistical confidence
desired. All methods for estimating 11)50 values require that
the test animals be randomly assigned to dose groups. TVo rabbits
per sex per dose are recommended in most cases. If a toxicolog-
ical effect occurs with a marginally significant incidence, data
from further testing with larger numbers of animals may be
required.
3. Females: The females should be nonpregnant since
pregnancy may modify response.
4. Dose levels: To establish a dose regimen, a trial
72
-------
test is recommended. It should include one dose level higher than
the expected ID$Q and at least one dose level below the
expected 11)50 . J^ a ^°se °f 2g/kg (or 2ml/kg) or more, placed
on the abraded (within two hours prior to application) skin of at
least 2 animals per sex, produces no mortality, no further testing
at other dose levels is necessary. However, if mortality occurs,
at least three dose levels should be used to estimate the
' using rabbits with intact skin.
5. Preparation of skin: Twenty-four hours before
testing, fur from the trunk of animals must be clipped so that no
less than 10% (about 240 cm-*) of the dorsal body surface area is
available for application of material. The abraded area is
prepared by making four epidermal incisions with a clean needle
through the stratum corneun, but not deep enough to disturb the
derma or produce bleeding.
B. Test Procedure
1. Test substance: When testing solids, the test
substance should be moistened sufficiently with normal saline or
tap water to make a paste that will insure good contact with the
skin. For some applications, it may be appropriate or necessary
73
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to use other vehicles. If a carrier or diluent is used, it should
be non-irritating and of known low toxicity. When such vehicles
are used, consideration should be given to the effects of those
vehicles on absorption of the test substance.
2. Dosage: When technically feasible, the maximum
quantity of substance plus vehicle to be applied is 2 gAg body
weight. The test substance should be applied uniformly over at
least 10% of the dorsal surface area. When possible, at least 3
levels of exposure should be tested to permit development of a
dose-response trend.
3. Administration (application): The test substance
must remain in contact with the skin throughout the exposure
period of 24 hours. Liquid or solid substances should be held in
contact with the skin with a porous gauze dressing and non-
irritating tape. The test site should be covered in a semi-
occlusive fashion with an impermeable material such as plastic
film or rubberized cloth.
Routine use of occlusive dressings is not recom-
mended. Occlusive skin dressings may enhance penetration of the
test substance and should be used only when testing for effects
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that may occur under similar conditions in humans.
During exposure, animals should be prevented from
ingesting or inhaling the test substance. Restrainers, such as
Elizabethan collars, that permit animals to move about their cages
should be used for this purpose. Immobilization is not a
recommended method.
At the end of the exposure period, all residual
material should be removed by washing, using an appropriate
solvent. About one half hour later, and once again at 72 hours,
the exposed area should be examined, and all lesions noted and
graded (Table I).
4. Observation period: The observation period must be
at least 14 days. However, duration of observation should not be
fixed; rather, it should be determined by the toxic reactions,
rate of onset, and length of recovery period. Although a 14-day
observation period is sufficient for most compounds, animals
demonstrating visible signs of toxicity after 14 days could be
held longer.
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5. Recording of clinical observations: The animals
should be observed frequently during the first day and twice a day
thereafter at least 4 hours apart (once each morning and late
afternoon). Observations should be recorded systematically as
they are made, and individual records should be maintained for
each animal. Observations should include, but not be limited to,
grossly visible changes in skin and fur, eyes, mucous membranes
and respiratory, cardiovascular, autonomic and central nervous
systems, and somatomotor activities. Particular attention should
be directed to observations for the presence of tremors, convul-
sions, salivation, diarrhea, lethargy, sleep, and coma. The time
at which toxicity signs appear and the time of death must be
recorded.
6. Weight change: Individual weights of animals must
be determined on the day the test substance is administered,
weekly thereafter, and at death or sacrifice.
6. Necropsy: A complete gross necropsy should be
performed on all animals that die during the course of the test
and on all remaining animals at termination of the test. Gross
pathological changes of the intestinal tract and the major organs
such as liver, kidney, heart, brain, and spleen should be noted.
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Liver, skin/ kidney/ and organs showing evidence of gross path-
ology of all animals surviving 12 or more hours should be
preserved for possible future microscopic examination.
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TABLE I
EVALUATION OF SKIN REACTION
Value
Erythema and Eschar Formation
No erythema 0
Very slight erythema (barely perceptthl.e) I.
Well-defined erythema , 7.
Moderate to sever erythem , ,3
Severe erythema (beet readness) to slight
eschar formation (injuries in depth) 4
Edema Formation
No edema , 0
Very slight edema (barely perceptible) ,, ..,,,,I
Slight edema (edges of area well defined by
definite raising .2
Moderate edema (raised approximately 1 milliliter) 3
Severe edema (raised more than 1 millimeter and
extending beyond the area of exposure) 4
Severe eschar and/or corrosion Note
occurence
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III. Data Reporting
A. Identification
Each test report must be signed by the person
responsible for the test and identify:
1. The laboratory where the test was performed by
name and address;
2. The inclusive dates of the test; and
3. Each person primarily responsible for separate
components of the test and the component for which the person is
reponsible including (a) the conduct of the test, (b) analysis of
the data/ (c) the writing of the report, and (3) any written or
other matter contained in the report.
B. Body of Report
The test report must include all information necessary
to provide a complete and accurate description and evaluation of
the test procedures and results. Each report must include the
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following sections:
1. Summary and Conclusions. This section of the test
report should contain a tabular summary of the data, an analysis
of the data, and a statement of the conclusions drawn from the
analysis. The summary must highlight all positive data or
observations and any deviations from control data which may be
indicative of toxic effects.
2. Materials. This section of the test report shall
include, but not be limited to, the following information:
(a) Identification of the test substance,
including:
i. chemical name, molecular structure, and a
qualitative and quantitative determination of its chemical
composition, including names and quantities of known contaminants
and impurities, so far as is practical; the determinations shall
also include a listing of materials as unknowns, if any, so that
100% of the test sample is acccounted for:
ii. manufacturer and lot number of the substance
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tested, and such information as physical state, pH, stability, and
purity; and
iii. exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.
(b) Animal data, including:
i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;
ii. source of supply of the animals, diet (lot
number, composition, etc.), and water;
iii. description of any pre-test conditioning;
iv. description of the method used in
randomization of animals to test or control groups; and
v. numbers of animals of each sex in each test
and control group.
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(c) Data on facilities should include description
of the caging conditions including number of animals per cage/
bedding material, ambient temperature, and humidity.
3. Methods
(a) Deviation from guidelines - This section
shall indicate all ways in which the test procedure deviates from
these guidelines and shall state the rationale for such
deviation.
(b) Specification of test methods - This section
shall include a full description of the experimental design and
procedure, the length of the study, and the dates on which the
study began and ended.
(c) Statistical analysis - All statistical methods
used should be fully described or identified by reference.
(d) Data on dosage administration, including:
i. all dose levels administered, expressed as
mg/kg of body weight;
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ii. method and frequency of administration; and
iii. total volume of substance (i.e./ test
substance plus vehicle) contained in individual dosages.
(e) Data on obsevation methods/ including:
i. duration; and
ii. method and frequency of observation of the
animals.
4. Results
The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.
(a) Tabulation of the response data (i.e./ number
of animals dying; number of animals showing signs of toxicity;
number of animals exposed) at each exposure level by sex/ and time
of death after dosing;
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(b) ID$Q values for each test substance
calculated at the end of the observation period, with method of
caluclation specified;
(c) 95% confidence interval for the
values;
(d) Slope of the dose-mortality curve for each
substance tested; and
(e) Findings from all clinical observations,
necropsy, and histopathological examinations (when made).
5. References
This section of the test report shall include the
following information:
(a) Availability of original data, specimens and
samples of the test substance. The location of all original data,
specimens, and samples of the test substances which are retained
in accordance with the testing requirement.
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(b) Literature or references, including/ where
appropriate, those references for (1) test procedures, (2)
statistical and other methods used to analyze the data, (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
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IV. Suggested Reading
1. Draize, J. H. 1959. Dermal toxicity. In; Appraisal of
the Safety of Chemicals in Poods, Drugs, and Cosmetics. Austin,
Texas. Association of Pood and Drug Officials of the U. S. pp.
46-59.
2. National Academy of Sciences - National Research
Council, 1977. Dermal and eye toxicity tests. In; Principles and
Procedures for Evaluating the Toxicity of Household Substances,
Report No. 1138, prepared for the Consumer Product Safety
Commission, pp. 23-28.
3. McCreesh, A. H. and M. Steinberg, 1977. Dermato-
Toxicology and Pharmacology, Hemisphere Publishing Corp.,
Washington, D. C.
4. Mailbach, H. I. and P. N. Marzulli. 1975. Animal Models
in Dermatology. Churchill and Livingston, Edinburgh.
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DRAFT I.R.L.G. GUIDELINE FOR
ACUTE INHALATION TESTS IN RATS
Testing Standards § Guidelines Work Group
INTERAGENCY REGULATORY LIAISON GROUP
June 6, 1979
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PREFACE
The purpose of this test is to characterize the toxicity of a sub-
stance administered acutely to test animals. This characterization
goes beyond simply counting dead animals or calculating LCso
values and includes clinical observation, identification of target
organs, etc.
Careful consideration was given to inhalation tests and techniques
which assess acute injuries to the lungs and systemic effects. This
guideline is for acute inhalation studies using rats in dynamic
airflow chambers, and it may be used, with minor changes, for other
species of rodents.
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Scientific Issues for Public Comment
During the development of this proposed guideline/ many scientific
issues were discussed by the Work Group. These issues were raised by
members of the Work Group, by the public comments to the EPA proposed
pesticide guidelines and by the comments of interagency reviewers.
Consideration of these comments, discussed below/ is reflected in this
proposed guideline; and the public is invited to comment further on
these issues or any other aspect of this proposed guideline.
A. Although commentors have suggested that inhalation effects may
not be sex dependent and that either sex may be used or if there is a
sex difference in the response to the test substance, the difference
will appear in the acute oral test, the Vtork Group thinks that both
males and females should be used in the acute inhalation test. Any
comments or information about which sex/ if either, is more appropriate
for an acute inhalation study and data showing sex differences or lack
of differences would be helpful to the Work Group.
B. This guideline requires no further acute inhalation testing
if an exposure to 5 mg/1 of the test substance for four hours produces
no mortality. Some commmentors pointed out the problems associated
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with achieving an exposure concentration of 5 mg/1. The Work Group
recognizes this problem and in this guideline has allowed for the
physical, chemical properties to be a determining factor in setting the
maximum dose.
C. While there are various opinions on the need for controls
(vehicle, sham, negative)in the acute inhalation study, the Work Group
thinks control groups are unnecessary in this test. When a solvent of
uncharacterized toxicity is used, however, an acute study should be
done on the solvent.
D. Temperature and humidity ranges for acute inhalation
toxicity testing were another issue. Lower or higher temperature
ranges were suggested, as well as no temperature limits. The Work Group
decided that temperature and relative humidity measurements were
necessary and that a temperature of 22°C + 2°C, with a relative
humidity of 30% to 50% would be appropriate for the conduct of this
test. One consideration in this decision was the fact that scientific
literature indicates that both these variables should be maintained
within relatively narrow ranges, since changes in either direction can
alter the toxic responses of the test substance.
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D. In view of the primary importance of the lungs in an
•
inhalation study, special, specific treatment of lungs prepratory to
histopathological examination was recommended. The Work Group
acknowledges the importance of special treatment of the lungs in an
acute inhalation toxicology study, but thinks there are several
acceptable procedures for preserving lung tissue, and decided to allow
for individual investigator judgment on this matter.
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TABLE OF CONTENTS
I. General Considerations
A. Good Laboratory Practices
B. Personnel
C. Test Substance
D. Animals
E. Dead Animals, Necropsy, and Histopathology
F. Equipment
II. Specific Considerations
A. Test Preparation
B. Test Procedure
III. Data Reporting
A. Identification
B. Body of Report
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1. Summary and Conclusions
2. Materials
3. Methods
4. Results
5. References
IV. Suggested Reading
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I. General Considerations
A. Good Laboratory Practices
Basic standards presented here relating to good laboratory
practices are to serve as general guidance for the conduct of the
study, but are not intended to be all inclusive. This guideline does
not set forth the managerial aspects of science or good laboratory
practices. Studies should be conducted according to "Nonclinical
Laboratory Studies, Good Laboratory Practice Regulations," (43 FR
59986, 22 December 1978).
B. Personnel
All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures. The agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing. Tb the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
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tency of evaluation. When a histopathological examination is
done, similar considerations should apply.
C. Test Substance (materials or mixtures of substances or
materials)
1. As far as is practical/ composition of the test
substance must be known, including the name and quantities of
known contaminants and impurities. Unknown materials, if any,
must be quantified to account for 100% of the test sample. The
specific substance to be tested will be determined in consultation
with each agency.
2. The lot of the substance tested should be the same
throughout the study. The test sample should be stored under
conditions that maintain its stability, strength, quality, and
purity from the date of its production until the tests are
complete.
3. Safe handling and disposition of the test substance
is essential.
D. Animals
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1. Animals used for testing should not have been sub-
jected to any previous experimental procedures.
2. The test animal shall be characterized as to species,
strain, sex, weight and/or age. Each animal must be assigned an
appropriate identification number.
3. Recommendations contained in DHEW pub. no. (NIH) 74-23,
entitled "Guide for the Care and Use of Laboratory Animals," should be
followed for the care, maintenance, and housing of animals.
4. Animals may be group-caged for this test unless pharma-
cological action of the test substance dictates otherwise. However,
the number of animals per cage should not prevent continued and clear
observation of each animal. When signs of morbidity or excitability
are observed in group-caged animals during the test, such animals
should be moved to separate cages.
5. Healthy animals must be used. Animals must be
assigned to groups in such a manner as to minimize bias and assure
comparability of pertinent variables.
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6. Each animal must be observed as necessary to insure
that animals are not lost due to cannibalism, autolysis of tissues,
misplacement, and similar management problems.
E. Dead Animals, Necropsy, and Histopathology
When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately. Necropsy must be performed with-
in 16 hours of death. When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopathological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.
F. Equipment
All equipment used in conducting the test, including equip-
ment used to prepare and administer the test substance and equipment
used to maintain environmental conditions, must be of appropriate
design and adequate capacity. Equipment should be inspected, cleaned,
and maintained regularly. The equipment must be properly calibrated at
the time of its use.
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II. Specific Considerations
A. Test Preparation
1. Animals: Standard laboratory strains of healthy young,
adult rats weighing between 125 and 250 g should be used, but other
species or younger animals may be required for specific purposes. For
example, immature animals must be used when attempting to estimate
LCso values that may apply to infants; and these studies should be
performed in addition to the studies on mature animals.
2. Number and sex: The number of animals must be adequate
for analysis. At least 10 animals, 5 per sex, should be used at each
concentration level. Also, if sex differences are seen in LC50
values, the study should be repeated using 10 of each sex. If a
toxicological effect occurs with a marginally significant incidence,
data from further testing with larger numbers of animals may be
required.
3. Females: Since estrus and pregnancy may modify female
responses, the females should be nulliparous and nonpregnant.
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4. Dosage concentration: To establish a regimen, a trial
test is recommended. It should include at least one concentration
level higher and one concentration lower than the expected LC$Q.
No further testing is necessary if an exposure of 5 mg/1 (or a maximum
concentration as permitted by the physical/chemical properties of the
test substance) for the prescribed 4 hour duration administered to 5
male and 5 female test animals produces no mortality. If mortality
occurs, however, an additional test using at least 4 concentration
levels and a negative control group should be done. The doses should
be spaced appropriately to produce test groups with mortality rates of
1-20%, about50%, and 70-99% permiting the calculation of the IC$Q
value with a 95% confidence interval of +_ 20% or less.
5. Use of solvent: If necessary to help generate an
appropriate concentration of the substance in the atmosphere, a solvent
may be added to the test substance. If the product's labeling instruc-
tions specify use of a particular solvent, that solvent is recommended.
If no solvent is specified in the product's labeling instructions, the
solvent(s) in the product formulation should be used if possible. If a
vehicle or solvent of uncharacterized toxicity is used in generating
the exposure atmosphere, an acute inhalation test should be done on the
solvent.
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6. Selection of equipnent: The animals should be tested
using inhalation equipnent designed to sustain a dynamic airflow and an
evenly distributed exposure atmosphere. If a chamber is used, its
design should minimize crowding of the test animals and maximize their
exposure to the test substance. References to examples of accceptable
experimental designs appears in Section IV, "Suggested Reading."
B. Test Procedure
1. The chamber should be maintained at 22°C +_ 2°, and
the relative humidity should be 30% to 50%.
2. Air flow should be adjusted to insure that the oxygen
content of exposure atmosphere is at least 19% and concentrations of
the test substance in the chamber at the outlet and the inlet are
essentially the same.
3. Monitoring or measurements shall be made of:
(a) the rate of airflow, continuously;
(b) the actual concentration of the test substance by
sampling chamber air as near as practical to the animals' breathing
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zone as frequently as necessary to obtain an average, integrated
external exposure which is representative of the entire exposure
period. Concentration and particle size distributions of the test
substance in the chamber should be controlled. During the development
of the generating system, particle size analysis should be carried out
as frequently as necessary to insure proper stability of aeresol
particles. During exposure, analysis should be made as often as
necessary to determine the consistency of particle distribution (at
least 20% of the particles should be 10 microns or less in diameter).
(c) the temperature and humidity, continuously.
4. The exposure period (duration of compound administra-
tion) shall be 4 hours.
5. The observation period must be at least 14 days. Dura-
tion of observation should not be fixed; it should be determined by the
toxic reactions, rate of onset, and length of recovery period. Although
a 14-day observation period is sufficient for most compounds, animals
demonstrating visible signs of toxicity after 14 days could be held
longer.
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6. Recording of clinical observations: The animals should
be observed frequently during the first day and twice a day thereafter
at least 4 hours apart (once each morning and late afternoon). Obser-
vations should be recorded systematically as they are made, and indi-
vidual records should be maintained for each animal. Visual observa-
tions should include, but not be limited to, changes in skin and fur,
eyes, mucous membranes and respiratory, cardiovascular, autonomic and
central nervous sytems, and somato-motor activities. Particular
attention should be directed to observations for the presence of
tremors, convulsions, salivation, hyperactivity, diarrhea, lethargy,
sleep, coma, blanching, cyanosis, and vasodilation. The time at which
toxicity signs appear and the time of death must be recorded.
7. Weight change: Individual weights of animals must
be determined on the day the test substance is administered,
weekly thereafter, and prior to sacrifice.
8. Necropsy: A complete gross necropsy should be per-
formed on all animals that die during the course of the test and all
remaining animals at termination of the test. Gross pathological
examination should include nasal passage, trachea, bronchi, lungs,
major organs of detoxification such as liver and kidneys, and any other
tissues known to be affected by the test substance. All abnormalities
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must be recorded. Lungs, liver, kidney, and organs showing evidence
of gross pathology of all animals surviving 12 or more hours should be
preserved for possible future microscopic examination.
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III. Data Reporting
A. Identification
Each test report must be signed by the person
responsible for the test and identify:
1. The laboratory where the test was performed by
name and address;
2. The inclusive dates of the test; and
3. Each person primarily responsible for separate
components of the test and the component for which the person is
reponsible including (a) the conduct of the test, (b) analysis of
the data, (c) the writing of the report, and (3) any written or
other matter contained in the report.
B. Body of Report,
The test report must include all information necessary
to provide a complete and accurate description and evaluation of
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the test procedures and results. Each report must include the
following sections:
1. Summary and Conclusions. This section of the test
report should contain a tabular summary of the data, an analysis
of the data, and a statement of the conclusions drawn from the
analysis. The summary must highlight all positive data or
observations and any other indications of toxic effects.
2. Materials. This section of the test report shall
include, but not be limited to, the following information:
(a) Identification of the test substance,
including:
i. chemical name, molecular structure, and a
qualitative and quantitative determination of its chemical
composition, including names and quantities of known contaminants
and impurities, so far as is practical; the determinations shall
also include a listing of materials as unknowns, if any, so that
100% of the test sample is acccounted for:
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ii. manufacturer and lot number of the substance
tested, and such information as physical state, pH, stability, and
purity; and
iii. exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.
(b) Animal data, including:
i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;
ii. source of supply of the animals;
iii. description of any pre-test conditioning,
including diet;
iv. description of the method used in
randomization of animals; and
v. numbers of animals of each sex in each test
group.
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(c) Data an facilities should include description
of the caging conditions including number of animals per cage,
inhalation chambers, bedding material, ambient temperature,
lighting conditions, and humidity.
3. Methods
(a) Deviation from guidelines - This section
shall indicate all ways in which the test procedure deviates from
these guidelines and shall state the rationale for such
deviation.
(b) Specification of test methods - This section
shall include a full description of the experimental design and
procedure, the length of the study, and the dates on which the
study began and ended.
(c) Statistical analysis - All statistical methods
used should be fully described or identified by reference.
(d) Data on dosage administration, including:
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i. all dose levels administered, including all
concentrations of substances expressed as milligrams/liter or
ii. method and frequency of administration; and
(e) Data on observation methods, including:
i. duration; and
ii. method and frequency of observation of the
animals.
(f) Data on equipment, including:
i. a description of the exposure chamber used
with justification for deviation from the design suggested in this
guideline;
ii. the rate of airflow through the chamber
(liters per minute);
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iii . gas measurements and analysis for the test
compound; and
iv. the method used in determining particulate
size and the results of the analysis.
4. Results
The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.
(a) Tabulation of the response data (i.e., number
of animals dying; number of animals showing signs of toxicity;
number of animals exposed) at each exposure level by sex, and time
of death after dosing;
(b) 1X50 values for each test substance
calculated at the end of the observation period, with method of
caluclation specified;
(c) 95% confidence interval for the
values;
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(d) Slope of the dose-mortality curve for each
substance tested; and
(e) Findings from all clinical observations,
necropsy, and histopathological examinations (when made).
5. References
This section of the test report shall include the
following information:
(a) Availability of original data, spec linens and
samples of the test substance. The location of all original data,
specimens, and samples of the test substances which are retained
in accordance with the testing requirement.
(b) Literature or references, including, where
appropriate, those references for (1) test procedures, (2)
statistical and other methods used to analyze the data, (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
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IV. Suggested Reading
1. Drew, R. R., and S. Laskin. 1973. Environment inhalation
chambers. In; Methods of Animal Experimentation. W. I. Gay, ed.
Academic Press, New York. Vol. 4, pp. 1-41.
2. Fraser, D. C., R. E. Bales, M. Lippmann, and H. E.
Stockinger. 1959. Exposure chamber in research in animal inhalation.
Public Health Monograph No. 57, U. S. Public Health Service, Department
of Health, Education and Welfare.
3. National Academy of Sciences - National Research Council.
1977. Inhalation exposure. In; Principles and Procedures for
Evaluating the Toxicity of Household Substances, Report No. 1138.
Washington, D. C. 4:60.
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DRAFT I.R.L.G. GUIDELINE FOR
TERATOGENICITY TESTING IN RAT,
MOUSE AND RABBIT
Testing Standards § Guidelines Work Group
INTERAGENCY REGULATORY LIAISON GROUP
May 16, 1979
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PREFACE
This guideline is for use with substances given orally to the rat,
mouse, or rabbit.
The purpose of this test is to yield data to help determine
whether a test substance is potentially embryotoxic and/or terato-
genic. Treatment must be started early enough and continued long
enough to include the period of organogenesis for the particular
species used.
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Scientific Issues for Public Comment
During the development of this proposed guideline, many scientific
issues were discussed by the Work Group. These issues were raised by
members of the Work Group, by the public comments to the EPA proposed
pesticide guidelines and by the comments of interagency reviewers.
Consideration of these comments, discussed below, is reflected in this
proposed guideline; and the public is invited to comment further on
these issues or any other aspect of this proposed guideline.
A. In this proposed guideline, dosing begins after implantation
and continues through organogenesis up to one day prior to term, which
could be Day 6 through 19 in the rat, or Day 7 through 29 in the rabbit
(depending upon the strain used). Since the purpose of this test is to
determine the teratogenicity of a substance, the Work Group believes
that implantation of the embryo should occur before dosing begins in
order to assure that the dosing will not interfere with implantation.
Also, this guideline provides that dosing will occur through most of
the period of gestation, which will in most species, go beyond the
period of organogenesis.
Comment on the duration of dosing relative to the gestation
period is encouraged.
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B. Some ooramentors suggested that doses should be adjusted peri-
odically throughout pregnancy rather than basing the dosage level on
the dam's weight on the first day of compound administration (Day 6 of
pregnancy in the rat and mouse, and Day 7 in the rabbit). Because of
the lack of evidence of the transplacental movement of the test
substance, and the uncertainty of escalating the maternal concentra-
tion relative to each dose level, the work group believes that dosage
should be based on the maternal weight just after implantation.
Additional information regarding the setting of dosage levels
would be helpful.
C. Another issue is the proper selection of dosages to be tested.
The highest dose suggested was one that either causes overt maternal
toxicity or affects fetal development. Because excessive maternal
intoxication may indirectly prevent normal fetal development, care
must be taken in choosing this dose. Hie main purpose of a teratology
study is to evaluate the potential of a chemical to affect fetal
development, and to produce anomalies in the offspring. Therefore,
the highest dose should not cause so many fetal deaths that the
assessment of its teratogenic potential is not possible. Some
commentors have suggested that the maximum dose should be limited to
10,000 times the expected human dose or somehow otherwise limited when
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testing relatively inert substances. The Work Group recognizes that
human exposure may not be known, constant, or measurable. Also, the
Work Group is unaware of a method for defining the maximum amount to
be given, except by either observing toxicity or by the limit of the
physical/chemical properties of the substances.
The public is encouraged to comment on the proper selection of
dose levels for teratology testing.
D. The appropriate number of pregnant animals per group is
another issue. Government agencies have traditionally requested that
at least 20 pregnant rats or mice and at least 10 pregnant rabbits be
used in each group. Recently other governments have asked for at
least 20 pregnant rabbits. The National Academy of Science recently
suggested that at least 20 pregnant animals, regardless of specie, be
used in each group. Although the Work Group supports the use of 20
pregnant rodents per group, it does not endorse the use of 20 pregnant
rabbits per group because of limited supplies of healthy rabbits and
the costs involved. The Work Group questions whether the additional
animals produce sufficient data to warrant the difficulties and
suggests the use of 15 pregnant rabbits per group as a compromise.
The public is encouraged to comment on the proper number of
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pregnant animals per group in teratology studies. Of particular value
would be comments on statistical or scientific rationale for determin-
ing the proper number of pregnant animals per group.
E. The necessity for positive control groups in a teratology
study has been questioned. This guideline recommends positive
controls to assure that the strain and species being used is sensitive
to known teratogens and that those conducting the studies are thor-
oughly familiar with identification of terata. Some ccranentors have
suggested that historical data may be sufficient for these purposes.
Others have suggested that positive controls should be used to char-
acterize the strain or species being used and that positive control
studies are necessary only when a laboratory selects a new or
different species or strain for use.
The Work Group would appreciate receiving comments on ways to
assure that the species being used are characterized as to their
ability to respond to known teratogens and to assure that individuals
examining offspring have had experience detecting a wide variety of
terata.
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TABLE OF CONTENTS
I. General Considerations
A. Good Laboratory Practices
B. Personnel
C. Test Substance
D. Animals
E. Dead Animals, Necropsy, and Histopathology
F. Equipment
II. Specific Considerations
A. Test Preparation
B. Test Procedure
III. Data Reporting
A. Identification
B. Body of Report
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1. Suiroary and Conclusions
2. Materials
3. Methods
4. Results
5. References
IV. Suggested Reading
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I. General Considerations
A. Good Laboratory Practices
Basic standards presented here relating to good labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive. This guide-
line does not set forth the managerial aspects of science or good
laboratory practices. Studies should be conducted according to
"Nonclinical Laboratory Studies, Good Laboratory Practice
Regulations," (43 PR 59986, 22 December 1978).
B. Personnel
All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures. Bie agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing. To the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
tency of evaluation. When a histopathological examination is
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done, similar considerations should apply.
C. Test Substance (materials or mixtures of substances or
materials)
1. As far as is practical/ composition of the test
substance must be known/ including the name and quantities of
known contaminants and impurities. Unknown materials/ if any,
must be quantified to account for 100% of the test sample. The
specific substance to be tested will be determined in consultation
with each agency.
2. The lot of the substance tested should be the same
throughout the study. The test sample should be stored under
conditions that maintain its stability/ strength/ quality/ and
purity from the date of its production until the tests are
complete.
3. Safe handling and disposition of the test substance
is essential.
D. Animals
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1. Animals used for testing should not have been sub-
jected to any previous experimental procedures.
2. The test animal shall be characterized as to
species, strain, sex, weight and/or age. Each animal must be
assigned an appropriate identification number.
3. Recommendations contained in DHEW pub. no. (NIH)
74-23, entitled "Guide for the Care and Use of Laboratory
Animals," should be followed for the care, maintenance, and
housing of animals.
4. Animals may be group-caged unless the pharmacolog-
ical action of the test substance dictates otherwise. However,
the number of animals per cage should not prevent continued and
clear observation of each animal. When signs of morbidity or
excitability are observed in group-caged animals during the test,
such animals should be moved to separate cages.
5. Healthy animals must be used. Animals must be
assigned to groups in such a manner as to minimize bias and assure
comparability of pertinent variables.
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6. Bach animal must be observed as necessary to insure
that animals are not lost due to cannibalism, autolysis of
tissues, misplacement, or similar management problems.
7. When control animals are used, they must be housed,
fed, and handled exactly like the test animals; and they must be
caged to minimize airborne or other contamination by the test
substance.
E. Dead Animals, Necropsy, and Histopathology
When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately. Necropsy must be performed with-
in 16 hours of death. When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopatnological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.
F. Equipment
All equipment used in conducting the test, including
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equipment used to prepare and administer the test substance and
equipment used to maintain environmental conditions, must be of
appropriate design and adequate capacity. Equipment should be
inspected, cleaned, and maintained regularly. The equipment must
be properly calibrated at the time of its use.
II. Specific Considerations
A. Test Preparation
1. Animals: Strains with low fecundity should not be
used. All test and control animals must be young, mature, pregnant
females of uniform age, size, and parity. Untreated males of
proven fertility should be used to produce the pregnancies.
2. Test groups: At least three test groups and one
vehicle control group must be used. When the test substance is
administered in a vehicle, the vehicle only should be administered
to the controls. If no vehicle is used, then the controls should
be sham treated. If there are insufficient data on the toxic
properties of the vehicle used in administering the test sub-
stance, a sham control group should also be included. In all
other respects, the controls must be handled and maintained in a
manner identical to that used with the groups given the test
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substance. For quality assurance, either a positive control group
of at least 5 pregnant animals should be included in every study
or a positive control group of 20 pregnant animals should be
included in a study at least once a year and when the species or
strain of animals being studied is changed. Any known teratogen
may be used as the positive control. Examples include aspirin or
Vitamin A for rats, 6 Amino nicotinamide for rabbits, and
corticosteroids for mice.
3. Number of animals: Sufficient numbers of anijnals
must be bred to assure that each test group and the vehicle
control group will consist of at least 20 pregnant rats or mice,
or at least 15 pregnant rabbits. These are the minimum numbers of
pregnant animals at or near term. The objective is to assure that
sufficient pups are produced to permit evaluation of the terato-
genic potential of the substance. As mentioned above, the posi-
tive control groups should routinely consist of at least 5
pregnant animals.
B. Test Procedure
1. Duration of test and time of delivery: Day 0 is
defined as the day a vaginal plug and/or sperm are found. The test
substance should be administered daily beginning soon after
implantation (Day 6 for rats or mice, Day 7 for rabbits) and con-
tinuing through most of the gestation period until about one day
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prior to term. Thus, the treatment period for the rat would be
Day 6 through Day 19 of pregnancy; for the rabbit it would be Day
7 through 29 of pregnancy; and in the mouse, the period would be
Day 6 until one day before expected delivery. In the mouse, the
period of gestation varies with the strain used.
For substances that cause enzyme induction, or are
highly toxic, shorter dosage periods may be appropriate.
In all cases, fetuses shall be delivered by
hysterotony about one day prior to term.
2. Dosage: At least three dosage levels must be
tested in addition to the controls. Unless limited by the
physical/chemical nature, or biological effects of the compound,
the highest dosage level should induce overt maternal toxicity or
affect fetal development. Maternal toxicity should not be so
great as to compromise the integrity of tlie study or obscure
meaning of the malformations. The intermediate dose(s) should
induce some observable fetal effects attributable to the test
substance. The low dosage level should not induce observable
adverse effects attributable to the test substance. The dosage
administered should be based on the individual animal's body
weight on the first day of substance administration.
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3. Route of Administration: The test substance or
vehicle should be administered by oral intubation unless the
chemical or physical characteristics, or pattern of human exposure
to the test substance suggest a more appropriate route of admin-
istration. The test substance should be administered at approxi-
mately the same time each day.
4. Animal care: Pood and water should be provided ad
libitum. Pregnant females may be provided nesting materials,
although it is not considered necessary.
5. Observation: Throughout the test period, each
animal must be observed at least once daily, by an appropriately
trained observer. Pertinent behavioral changes, and all signs of
toxicity, including mortality, must be recorded. Any female
showing signs of abortion or premature delivery must be sacrificed
on the data such signs are observed. These observations should be
reported individually. Females should be weighed at the start of
substance administration (Day 6 or 7), at the time of sacrifice,
and at least weekly between these times.
6. Necropsy: Immediately after a female is sacri-
ficed, the uterus should be excised and examined for embryonic or
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fetal deaths and the lumber of live fetuses. When possible, the
tune of death in utero should be established. The fetuses should
be examined externally, weighed individually, and the weights
recorded. The sex of each fetus should be determined if possible.
In rodents about one half of each litter should be eviscerated,
prepared, and examined for skeletal anomalies using the method of
Staples (11) or equivalent. The remaining one half of each litter
should be prepared and examined for soft tissue anomalies, using
the method of Wilson (15) or equivalent. In rabbits, all fetuses
should be examined by gross dissection for soft tissue anomalies
and subsequently processed for skeletal examinations.
7. Statistical Analysis: Values from the control and
test groups should be compared statistically. Any of several
methods are acceptable. The following are suggested: Anomalies
may be compared by chi-square methods or the binomial expansion
method. Maternal body weight gains and weight of fetuses may be
compared to those of controls by F-test and Student's t-test.
Petal survival and incidence of abnormalities per litter may be
compared by nonparametric, rank-order methods. Other statistical
methods may be substituted.
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III. Test Report
A. Identification
Each test report must identify:
1. The laboratory where the test was performed by
name and address;
2. The inclusive dates of the test; and
3. Each person primarily responsible for separate
components of the test and the component for which the person is
reponsible including (a) the conduct of the test, (b) analysis of
the data, (c) the writing of the report, and (3) any written or
other matter contained in the report.
B. Body of Report
The test report must include all information necessary
to provide a complete and accurate description and evaluation of
the test procedures and results. Each report must include the
following sections:
1. Summary and Conclusions. This section of the test
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report should contain a tabular summary of the data, an analysis
of the data, and a statement of the conclusions drawn from the
analysis. The summary must highlight all positive data or
observations and any deviations from control data which may be
indicative of toxic effects.
2. Materials. This section of the test report shall
include, but not be limited to, the following information:
(a) Identification of the test substance,
including:
i. chemical name, molecular structure, and a
qualitative and quantitative determination of its chemical
composition, including names and quantities of known contaminants
and impurities, so far as is practical; the determinations shall
also include quantitites of unknown materials, if any, so that
100% of the test sample is accounted for:
ii. manufacturer and lot number of the substance
tested, and such information as physical state, pH, stability, and
purity; and
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iii. exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.
(b) Animal data, including:
i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;
ii. source of supply of the animals;
iii. description of any pre-test conditioning,
including diet;
iv. description of the method used in
randomization of animals to test or control groups; and
v. numbers of animals of each sex in each test
and control group.
vi. parity
(c) Data on facilities should include description
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of the caging conditions including number of animals per cage,
bedding material, ambient temperature, humidity, and lighting
conditions.
3. Methods
(a) Deviation from guidelines - This section
shall indicate all ways in which the test procedure deviates from
these guidelines and shall state the rationale for such
deviation.
(b) Specification of test methods - This section
shall include a full description of the experimental design and
procedure, the length of the study, and the dates on which the
study began and ended.
(c) Statistical analysis - All statistical methods
used should be fully described or identified by reference.
(d) Data on dosage administration, including:
i. all dose levels administered, expressed as
mg/kg of body weight;
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ii. method and frequency of administration; and
iii. total volume of substance (i.e., test
substance plus vehicle) contained in individual dosages.
(e) Data on obsevation methods, including:
i. duration; and
ii. method and frequency of observation of the
animals.
4. Results
The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.
(a) Data on dose levels including the number of
animals initially on study, number and percentage that were
pregnant, number and percentage that died, and the average*
maternal body weights and all weight changes.
* All averages should be accompanied by an appropriate measure of
variability.
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(b) Maternal data for each animal should include
the following information arranged by group.
i. clinical signs of toxicity: a description
of all observed signs of toxicity accompanied by each animal's
identification number, test group and (i.e., day of pregnancy) of
observation,
ii. age (or weight) at the start of the
test,
iii. body weights on the first day of admin-
istration, at sacrifice, and at least once near mid-gestation; the
body weight change based on the carcass weight, i.e., body less
the uterus and its contents; and
iv. signs of resorptions, abortion, or
premature delivery.
(c) Fetal data: The following information
arranged by test group should be supplied.
i. cumulative data, showing mean and
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variability for each dose level: number of litters examined,
number of implantations per litter, average number of live pups
per litter, total of dead fetuses per litter, total number of
fetuses, number and percent of pups with anomalies, skeletal vs.
visceral anomalies, number and percent of litters containing
anomalous pups, and number and percent of abnormal pups par
litter.
ii. numerical data for each litter including:
identification number of each dam and/or its litter; number of
implantations; weight, number and percent of dead fetuses; number
and percent of live pups; average weight of live pups per litter;
when determined, number of each sex and percent of male pups;
number and percent of pups with any abnormality.
iii. anomaly data for each litter including:
identification number of the litter; number of pups examined;
number of pups having anomalies; and number of pups having
visceral anomalies. When an anomaly is difficult to describe,
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color photographs of anomalies may be submitted. If photographs
are taken, the equipment and film must be of sufficient quality to
permit controlled, close-up color photography of the anomaly to
yield clear, sharp-focus images that literally fill the camera
field.
(d) Evaluation of the results should
include:
i. an evaluation of the relationship, if
any, between exposure to the test substance and the anomalies,
and
ii. an indication of the dosage level at
which no toxic effects attributable to the test substance
appeared.
5. References
This section of the test report shall include the
following information:
(a) Availability of original data, specimens and
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samples of the test substance. The location of all original data/
specimens, and samples of the test substances which are retained
in accordance with the testing requirement.
(b) Literature or references, including, where
appropriate, those references for (1) test procedures, (2)
statistical and other methods used to analyze the data, (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
IV. Suggested Reading
1. Asling, C. W. 1969. Nutrition and teratogenesis.
Irj: Methods for Teratological Studies in Experimental Animals and
Man. H. Nishimara, J.R. Miller, and M. Vasuda, eds. Igaku-Shoin
Ltd.: Tokyo, pp. 76-92.
2. Collins, T. F. X. and E. V. Collins. 1976. Current
methodology in teratogenicity research. In; Advances in Modern
Toxicology. M. A. Mehlman (NIH), Vol. 1, Part 1, New Concepts in
Safety Evaluation, M. A. Mehlman, R. Shapiro, and H. Blumenthal.
Chapt. 6. Hemisphere Pub. Corp., Washington, D. C. pp. 144-174.
3. Perm. V. H. 1967. The use of the golden hamster in
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experimental teratology. Lab. Animal Care, 17:452.
4. Health Protection Branch. 1977. The Testing of
Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity.
Ministry of Health and Welfare: Canada.
5. Oser, B. L. 1971. Toxicology of pesticides to
establish proof of safety. In; Pesticides in the Environment.
Vol. 1, Part II, pp. 411-456. R. White-Stevens, ed. Marcel
Dekker, Inc.: New York.
6. Palludan, B. 1966. Swine in teratological studies.
In; Swine in Biochemical Research. L. K. Bustad and R. 0.
McClellan, eds. Battelle Mem. Inst.: Richland, Washington, pp.
51-68.
7. Palmer, A. K. 1974. Problems associated with the
screening of drugs for possible teratogenic activity. Exp.
Embryol. and Teratol. 1:16-33.
8. Robson, J. M. 1970. Testing drugs for teratogenicity
and their effects on fertility. Brig. Med. Bull. 26: 212-216.
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9. Bassoon, H. 1976. Survey and Evaluation of Techniques
Used in Testing Chemical Substances for Teratogaiic Effects. EPA
Report No. 560-5-77-007, NTIS Pub. PB-273-195. National
Technical Information Service: Springfield, Va.
10. Shepard, T. H., J. R. Miller and M. Morris. 1975.
Methods for Detecting of Environmental Agents that Produce
Congenital Defects. Proceedings of the Gaudeloupe Conference
Sponsored by 1'Institut de la Vie. North-Holland Publishing Col:
Amsterdam-Oxford: American Elsevier.
11. Staples, R. E. and Schnell, V. L. 1964. Refinements in
Rapid Clearing Technic in the KOH-Alizarin Red S Methods for Petal
Bone. Stain Tech. 29:61-63.
12. Tachmann Deplessis, G. 1972. Teratogenic drug
screening. Present procedures and requirements. Teratology.
5:271-285.
13. Weil, C. S. 1970. Selection of the valid number of
sampling units and a consideration of their combination in
toxicological studies involving reproduction, teratogenesis or
carcinogenesis. Food Cosmet. Toxicol. 8:177-182.
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14. Wilson, J. G. 1971. Use of rhesus monkeys in
teratological studies. Pad. Proc. Am. Soc. Exp. Biol.
30:104-109.
15. Wilson, J. G. 1973. Environmental Birth Defects.
Academic Press Inc. New York.
16. World Health Organization. 1967. Principles for the
testing of drugs for teratogenicity. WHO Tech. Rep. Ser. No. 364.
Geneva.
«U.S. GOVERNMENT PRINTING OFFICE ! 1979 0-300-043/6416
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