United States Prevention, Pesticides EPA712-C-96-202
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3250
Subchronic Dermal
Toxicity—90 days
"Public Draft'
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 870.3250 Subchronic Dermal Toxicity—90 days.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 798.2250 Dermal Toxicity;
OPP 82-3 90-Day Dermal (Pesticide Assessment Guidelines, Subdivision
F—Hazard Evaluation; Human and Domestic Animals) EPA report 5407
09-82-025, 1982; and OECD 411 Subchronic Deraml Toxicity: 90-Day.
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a chemical, the determination of Subchronic dermal toxicity may
be carried out after initial information on toxicity has been obtained by
acute testing. The subchronic dermal study has been designed to permit
the determination of the no-observed-effect level (NOEL) and toxic effects
associated with continuous or repeated exposure to a test substance for
a period of 90 days. This study is not capable of determining those effects
that have a long latency period for development (e.g., carcinogenicity and
life shortening). Extrapolation from the results of this study to humans
is valid only to a limited degree. It can, however, provide useful informa-
tion on the degree of percutaneous absorption, target organs, the possibili-
ties of accumulation, and can be of use in selecting dose levels for chronic
studies and for establishing safety criteria for human exposure.
(c) Definitions. The definitions in section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.
Cumulative toxicity is the adverse effect of repeated doses occurring
as a result of prolonged action or increased concentration of the adminis-
tered test substance or its metabolites in susceptible tissues.
Dose in a subchronic dermal study is the amount of test substance
applied daily to the skin for 90 days. Dose is expressed as weight of the
test substance (grams, milligrams), per unit body weight of test animal
(milligrams per kilogram), or as weight of the test substance per unit of
surface area (milligrams per square centimeter).
No-observed-effect level (NOEL) is the maximum dose used in a
study which produces no adverse effects. The NOEL is expressed in terms
of the weight of a test substance given daily per unit weight of test animal
(milligrams per kilogram per day).
Subchronic dermal toxicity is the adverse effects occurring as a result
of the repeated daily exposure of experimental animals to a chemical by
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the dermal route for a part (approximately 10 percent) of the test animal's
life span.
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures described for this guideline, produces
no observable toxic effects or if toxic effects would not be expected based
upon data on structurally related compounds, a full study using three dose
levels might not be necessary.
(e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian species should be used for testing. The rat, rabbit, or guinea
pig may be used. Commonly used laboratory strains should be employed.
If other mammalian species are used, the tester should provide justifica-
tion/reasoning for their selection. When a subchronic dermal study is con-
ducted as a preliminary to a chronic study, the same species and strain
should be used in both studies.
(ii) Age. (A) Testing should be started with young healthy animals
as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At the commencement of the study, the weight variation of ani-
mals used should not exceed + 20 percent of the mean weight for each
sex.
(iii) Sex. Equal numbers of animals of each sex with healthy skin
should be used at each dose level. The females should be nulliparous and
nonpregnant except for specially designed studies.
(iv) Numbers. (A) At least 20 animals (10 animals per sex) should
be used at each dose level.
(B) If interim sacrifices are planned, the number should be increased
by the number of animals scheduled to be sacrificed before completion
of the study.
(C) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(D) Each animal should be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
should be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals should be housed in individual cages.
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(B) The temperature of the experimental animal rooms should be at
22 + 3 °C.
(C) The relative humidity of the experimental animal rooms should
be 30 to 70 percent.
(D) Where lighting is artificial, the sequence should be 12 h light/
12 h dark.
(E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed to assure adequacy of nutritional require-
ments of the species tested and for impurities that might influence the
outcome of the test. Animals should be fed and watered ad libitum with
food replaced at least weekly. For nonrodents feeding should be at least
daily and water ad libitum.
(F) the study should not be initiated until animals have been allowed
a period of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine.
(2) Control and test substances, (i) Where necessary, the test sub-
stance is dissolved or suspended in a suitable vehicle. If a vehicle or dilu-
ent is needed, the vehicle should not elicit toxic effects or substantially
alter the chemical or toxicological properties of the test substance.
(ii) One lot of the test substance should be used, if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability. Prior to the initiation
of the study, there should be a characterization of the test substance, in-
cluding the purity of the test compound and if technically feasible, the
name and quantities of unknown contaminants and impurities.
(iii) If the test substance is dissolved or suspended in a vehicle, the
period during which the test substance is stable in such a mixture should
be determined prior to the initiation of the study. Its homogeneity and
concentration should be determined prior to the initiation of the study and
periodically during the study. Statistically randomized samples of the mix-
ture should be analyzed to ensure that proper mixing, formulation, and
storage procedures are being followed, and that the appropriate concentra-
tion of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This
group should be an untreated or sham-treated control group or, if a vehicle
is used in the application of the test substance, a vehicle control group.
If the toxic properties of the vehicle are not known or not available, both
untreated/sham-treated and vehicle control groups are required.
(4) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high dose level for 90 days and observed
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for reversibility, persistence, or delayed occurrence of toxic effects for a
post-treatment period of appropriate length, normally not less than 28 days.
In addition a control group of 20 animals (10 animals of each sex) should
be added to the satellite study.
(5) Dose levels and dose selection, (i) In subchronic toxicity tests,
it is desirable to determine a dose-response relationship as well as a
NOEL. Therefore, at least three dose levels plus a control and, where ap-
propriate, a vehicle control (corresponding to the concentration of vehicle
at the highest dose level) group should be used. Doses should be spaced
appropriately to produce test groups with a range of toxic effects. The
data should be sufficient to produce a dose-response curve.
(ii) The highest dose level should elicit signs of toxicity but not
produce severe skin irritation or an incidence of fatality which would pre-
vent a meaningful evaluation. If application of the test substance produces
severe skin irritation, the concentration may be reduced, although this may
result in a reduction in, or absence of, other toxic effects at the high dose
level. If the skin has been badly damaged early in the study, it may be
necessary to terminate the study and undertake a new one at lower con-
centrations.
(iii) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects.
(iv) The lowest dose level should not produce any evidence of toxic
effects.
(6) Preparation of animal skin. Shortly before testing, fur should
be clipped from not less than 10 percent of the body surface area for appli-
cation of the test substance. In order to dose approximately 10 percent
of the body surface, the area starting at the scapulae (shoulders) to the
wing of the ileum (hipbone) and half way down the flank on each side
of the animal should be shaved. Shaving should be carried out approxi-
mately 24 h before dosing . Repeated clipping or shaving is usually needed
at approximately weekly intervals. When clipping or shaving the fur, care
should be taken to avoid abrading the skin which could alter its permeabil-
ity.
(7) Preparation of test substance, (i) Liquid test substances are gen-
erally used undiluted, except as indicated in paragraph (e)(5)(ii) of this
guideline.
(ii) Solids should be pulverized when possible. The substance should
be moistened sufficiently with water or, when necessary, a suitable vehicle
to ensure good contact with the skin. When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account.
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(iii) The volume of application should be kept constant, e.g. less than
300 (j,L for the rat; different concentrations of test solution should be pre-
pared for different dose levels.
(8) Administration of test substance, (i) The duration of exposure
should be at least for 90 days.
(ii) The animals should be treated with test substance for at least 6
h/day on a 7-day per week basis. However, based on practical consider-
ations, application on a 5-day per week basis is acceptable. Dosing should
be conducted at approximately the same time each day.
(iii) The test substance should be applied uniformly over the treatment
site.
(iv) The surface area covered may be less for highly toxic substances.
As much of the area should be covered with as thin and uniform a film
as possible.
(v) During the exposure period, the test substance should be held in
contact with the skin with a porous gauze dressing. The test site should
be further covered with nonirritating tape to retain the gauze dressing and
the test substance and to ensure that the animals cannot ingest the test
substance. Restrainers may be used to prevent the ingestion of the test
substance, but complete immobilization is not recommended.
(9) Observation period. The animals should be observed for a period
of 90 days. Animals in the satellite group, (if used) scheduled for follow-
up observations should be kept for at least 28 days further without treat-
ment to assess reversibility.
(10) Observation of animals, (i) Observations should be made at
least once each day for morbidity and mortality. Appropriate actions
should be taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrifice of
weak or moribund animals).
(ii) A careful clinical examination should be made at least once week-
ly. Observations should be detailed and carefully recorded, preferably
using explicitly defined scales. Observations should include, but not be
limited to, evaluation of skin and fur, eyes and mucous membranes, res-
piratory and circulatory effects, autonomic effects such as salivation,
central nervous system effects, including tremors and convulsions, changes
in the level of motor activity, gait and posture, reactivity to handling or
sensory stimuli, grip strength, and stereotypies or bizarre behavior (e.g.,
self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed includ-
ing the time of onset, degree and duration.
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(iv) Individual weights of animals should be determined shortly be-
fore the test substance is administered, weekly thereafter, and at death.
(v) Food consumption should also be determined weekly if abnormal
body weight changes are observed.
(vi) Moribund animals should be removed and sacrificed when no-
ticed and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the treatment groups should be
sacrificed.
(11) Clinical pathology. Hematology and clinical chemistry examina-
tions should be made on all animals, including controls, of each sex in
each group at the end of the study.
(i) Hematology. The recommended parameters are: hemoglobin and
hematocrit concentrations, red blood cell count, white blood cell count,
differential leukocyte count, platelet count, and a measure of clotting po-
tential such as prothrombin time or thromboplastin time.
(ii) Clinical chemistry. Parameters which are considered appropriate
to all studies are electrolyte balance, carbohydrate metabolism, and liver
and kidney function. The selection of specific tests will be influenced by
observations on the mode of action of the substance and signs of clinical
toxicity. Suggested blood clinical chemistry determinations:
(A) Electrolytes.
(7) Calcium.
(2) Chloride.
(3) Magnesium.
(4) Phosphorous.
(5) Potassium.
(6) Sodium.
(B) Enzymes.
(7) Alkaline phosphatase.
(2) Alanine aminotransferase.
(3) Aspartate aminotransferase.
(4) Gamma glutamyl transferase.
(5) Sorbitol dehydrogenase.
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(C) Other.
(7) Albumin.
(2) Blood creatinine.
(3) Blood urea nitrogen.
(4) Globulin.
(5) Glucose (fasting).
(6) Total bilirubin.
(7) Total cholesterol.
(8) Total serum protein.
Other determinations which may be necessary for an adequate toxi-
cological evaluation include analyses of lipids, hormones, acid/base bal-
ance, methemoglobin and cholinesterase activity. Additional clinical bio-
chemistry may be employed where necessary to extend the investigation
of observed effects.
(iii) Urinalysis is not recommended on a routine basis, but only when
there is an indication based on expected OR observed toxicity.
(12) Optional immunotoxicity screen. To fulfill (in part) require-
ments for an immunotoxicity screen, subpopulation of splenic or peripheral
blood lymphocytes in the rodents should be enumerated and quantified.
Total T-, Total B-, Total T-helper, T-suppressor/cytotoxic and Natural
Killer (NK) cell populations should be determined on at least 10 rodents
of each sex in each group at the end of 90 days.
(13) Ophthalmological examination. Using an ophthalmoscope or
an equivalent device , ophthalmological examinations should be made on
all animals prior to the administration of the test substance and on all
high dose and control groups at termination. If changes in the eyes are
detected, all animals in the other dose groups should be examined.
(14) Gross necropsy, (i) All animals should be subjected to a full
gross necropsy which includes examination of the external surface of the
body, all orifices, and the cranial, thoracic and abdominal cavities and their
contents.
(ii) The liver, lungs, brain, kidneys, spleen, and gonads should be
trimmed and weighed wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples there-
of, should be preserved in a suitable medium for possible future
histopathological examination:
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(A) Digestive system.
(7) Salivary glands.
(2) Esophagus.
(3) Stomach.
(4) Duodenum.
(5) Jejunum.
(6) Ileum.
(7) Cecum.
(8) Colon.
(9} Rectum.
(10} Liver.
(11} Pancreas.
(12} Gallbladder (dogs) .
(B) Nervous system.
(1} Brain (multiple sections).
(2) Pituitary.
(3} Peripheral nerve(s).
(4) Spinal cord (three levels).
(5) Eyes (retina, optic nerve).
(C) Glandular system.
(1} Adrenals.
(2) Parathyroids.
(3} Thyroids.
(D) Respiratory system.
(1} Trachea.
(2} Lung.
(3} Pharynx.
(4) Larynx.
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(5) Nose.
(E) Cardiovascular/Hematopoietic system.
(7) Aorta (thoracic).
(2) Heart.
(3) Bone marrow.
(4) Lymph nodes.
(5) Spleen.
(6) Thymus.
(F) Urogenital system.
(7) Kidneys.
(2) Urinary bladder.
(3) Prostate.
(4) Testes.
(5) Epididymides.
(6) Seminal vesicle(s).
(7) Uterus.
(8) Ovaries.
(G) Other.
(7) All gross lesions and masses.
(2) Skin.
(3) Sternum and/or femur.
(15) Histopathology. (i) The following histopathology should be per-
formed:
(A) Full histopathology on the organs and tissues, listed under para-
graph (e)(14)(iii) of this guideline, of all animals in the control and high
dose groups.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to exam-
ination of the lungs of rodents should be made for evidence of infection
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as this provides a convenient assessment of the state of health of the ani-
mals.
(ii) If excessive early deaths or other problems occur in the high dose
group compromising the significance of the data, the next dose level
should be examined for complete histopathology.
(iii) An attempt should be made to correlate gross observations with
microscopic findings.
(iv) Tissues and organs designed for microscopic examination should
be fixed in 10 percent buffered formalin or a recognized suitable fixative
as soon as necropsy is performed and no less than 48 hours prior to trim-
ming. Tissues should be trimmed to a maximum thickness of 0.4 cm for
processing.
(v) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing toxic effects in the
treated groups.
(f) Data and reporting—(1) Treatment of results, (i) Data should
be summarized in tabular form, showing for each test group, number of
animals at the start of the test, the number of animals showing lesions,
the types of lesions and the percentage of animals displaying each type
of lesion.
(ii) All observed results, (quantitative and qualitative) should be eval-
uated by an appropriate statistical method. Any, generally accepted statis-
tical method should be used; the statistical methods including significance
criteria should be selected during the design of the study.
(2) Evaluation of study results. The findings of a subchronic dermal
toxicity study should be evaluated in conjunction with the findings of pre-
ceding studies and considered in terms of toxic effects and the necropsy
and histopathological findings. The evaluation should include the relation-
ship between the dose of the test substance, the incidence and severity
of abnormalities including behavioral and clinical abnormalities, gross le-
sions, identified target organs, body weight changes, effect on mortality,
and any other general or specific toxic effects. A properly conducted 90-
day subchronic dermal study should provide information on the effects
of repeated application of a substance and a satisfactory estimation of a
NOEL. It also can indicate the need for an additional longer-term study
and provide information on the selection of dose levels.
(3) Test report. In addition to reporting requirements specified under
EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92-
64-12367-9), the following specific information should be reported:
(i) Test substance characterization should include:
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(A) Chemical identification.
(B) Lot or batch numbers
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle if used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection
if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
(D) Identification of animal diet.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regime including levels, method, and volume.
(v) Test results should include:
(A) Group animal data: Tabulation of toxic response data by species,
strain, sex and exposure level for:
(7) Number of animals exposed.
(2) Number of animals showing signs of toxicity.
Number of animals dying.
(B) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(7) Date of death during the study or whether animals survived to
termination.
(2) Date of observation of each abnormal sign and its subsequent
course.
Body weight data.
(4) Feed consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
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(6} Results of hematological tests performed.
(7) Results of clinical chemistry tests performed.
(8) Results of immunochemistry screen, when performed.
(9) Necropsy findings, including absolute and relative organ weight
data.
(10) Detailed description of all histopathological findings.
(11) Statistical treatment of results, where appropriate.
(g) Quality control. A system should be developed and maintained
to assure and document adequate performance of laboratory staff and
equipment. The study must be conducted in compliance with GLP regula-
tions.
(h) References. The following references should be consulted for
background information on this test guideline:
(1) Draize, J.H. Dermal toxicity. Appraisal of Chemicals in Food,
Drugs and Cosmetics. The Association of Food and Drug Officials of the
United States (1959) 3rd printing 1975. pp. 46-59.
(2) National Academy of Sciences. Principles and Procedures for
Evaluating the Toxicity of Household Substances. A report prepared by
the Committee for the Revision of NAS Publication 1138, under the aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977).
(3) Organization for Economic Co-Operation and Development.
Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 411
Subchronic Toxicity Studies, Paris, 1981.
(4) United States Environmental Protection Agency. Office of Testing
and Evaluation. Proposed Health Effects Test Standards for Toxic Sub-
stances Control Act Test Rules. 40 CFR Part 772. Standard for Develop-
ment of Test Data. Subpart D. Federal Register. Vol. 44, No.91. Pp.
27350-27362.
(5) World Health Organization. Part I. Environmental Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals. (Ge-
neva: World Health Organization, 1978).
(6) World Health Organization. Guidelines for Evaluation of Drugs
for Use in Man, WHO Technical Report Series No. 563.(Geneva: World
Health Organization, 1975).
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(7) World Health Organization. Principles for Pre-Clinical Testing of
Drug Safety, WHO Technical Report Series No. 341.(Geneva: WHO,
1966).
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