[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9120]
[Page 326-330]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart H_Health Effects Test Guidelines
Sec. 799.9120 TSCA acute dermal toxicity.
(a) Scope. This section is intended to meet the testing requirements
under section 4 of the Toxic Substances Control Act (TSCA). In the
assessment and evaluation of the toxic characteristics of a substance,
determination of acute dermal toxicity is useful where exposure by the
dermal route is likely. It provides information on health hazards likely
to arise from short-term exposure by the dermal route. Data from an
acute study may serve as a basis for classification and labeling. It is
an initial step in establishing a dosage regimen in subchronic and other
studies and may provide information on dermal absorption and the mode of
toxic action of a substance by this route. An evaluation of acute
toxicity data should include the relationship, if any, between the
exposure of animals to the test substance and the incidence and severity
of all abnormalities, including behavioral and clinical abnormalities,
the reversibility of observed abnormalities, gross lesions, body weight
changes, effects on mortality, and any other toxic effects.
(b) Source. The source material used in developing this TSCA test
guideline is the Office of Prevention, Pesticides, and Toxic Substances
(OPPTS) harmonized test guideline 870.1200 (August 1998, final
guideline). This source is available at the address in paragraph (f) of
this section.
(c) Definitions. The following definitions apply to this section.
Acute dermal toxicity is the adverse effects occurring within a
short time of dermal application of a single dose of a substance or
multiple doses given within a 24-hour period.
Dosage is a general term comprising the dose, its frequency and the
duration of dosing.
Dose is the amount of test substance applied. Dose is expressed as
weight of test substance (grams, milligrams) per unit weight of test
animal (e.g., milligrams per kilogram).
Dose-effect is the relationship between the dose and the magnitude
of a defined biological effect either in an individual or in a
population sample.
Dose-response is the relationship between the dose and the
proportion of a population sample showing a defined effect.
LD50 (median lethal dose), dermal, is a statistically
derived estimate of a single dose of a substance that can be expected to
cause death in 50% of treated animals when applied to the skin. The
LD50 value is expressed in terms of weight of test substance
per unit weight of test animal (milligrams per kilogram).
(d) Approaches to the determination of acute toxicity. (1) EPA
recommends the following means to reduce the number of animals used to
evaluate acute effects of chemical exposure while preserving its ability
to make reasonable judgments about safety:
(i) Using data from substantially similar mixtures. In order to
minimize the need for animal testing, the Agency encourages the review
of existing acute toxicity information on mixtures that are
substantially similar to the mixture under investigation. In certain
cases it may be possible to glean enough information to make preliminary
hazard evaluations that may reduce the need for further animal testing.
(ii) Limit test. When data on structurally related chemicals are
inadequate, a limit test may be considered. If rodents are used, a limit
dose of at least 2,000 mg/kg bodyweight may be administered to a single
group of five males and five females using the procedures described in
paragraph (e) of this section. If no lethality is demonstrated, no
further testing for acute dermal toxicity is needed. If compound-related
mortality is produced,
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further study may need to be considered.
(2) [Reserved]
(e) Conventional acute toxicity test--(1) Principle of the test
method. The test substance is applied dermally in graduated doses to
several groups of experimental animals, one dose being used per group.
The doses chosen may be based on the results of a range finding test.
Subsequently, observations of effects and deaths are made. Animals that
die during the test are necropsied, and at the conclusion of the test
the surviving animals are sacrificed and necropsied. This section is
directed primarily to studies in either rats, rabbits, or guinea pigs
but may be adapted for studies in other species. Animals showing severe
and enduring signs of distress and pain may need to be humanely
sacrificed. Dosing test substances in a way known to cause marked pain
and distress due to corrosive or irritating properties need not be
carried out.
(2) Substance to be tested. Test, control, and reference substances
are discussed in 40 CFR Part 792--Good Laboratory Practice Standards.
(3) Test procedures--(i) Preparations. Healthy young adult animals
are acclimatized to the laboratory conditions for at least 5 days prior
to the test before the test animals are randomized and assigned to the
treatment groups.
(ii) Animal selection--(A) Species and strain. The rat, rabbit, or
guinea pig may be used. The albino rabbit is preferred because of its
size, ease of handling, skin permeability, and extensive data base.
Commonly used laboratory strains must be employed. If a species other
than rats, rabbits, or guinea pigs is used, the tester must provide
justification and reasoning for its selection.
(B) Age. Young adult animals, rats between 8- and 12-weeks-old,
rabbits at least 12-weeks-old, and guinea pigs between 5- and 6-weeks-
old at the beginning of dosing should be used. The weight variation of
animals used in a test must be within 20% of the mean weight for each
sex.
(C) Number and sex of animals. (1) At least five experimentally
naive animals with healthy intact skin are used at each dose level. They
should all be of the same sex. After completion of the study in one sex,
at least one group of five animals of the other sex is dosed to
establish that animals of this sex are not markedly more sensitive to
the test substance. The use of fewer animals may be justified in
individual circumstances. Where adequate information is available to
demonstrate that animals of the sex tested are markedly more sensitive,
testing in animals of the other sex may be dispensed with. An acceptable
option would be to test at least one group of five animals per sex at
one or more dose levels to definitively determine the more sensitive sex
prior to conducting the main study.
(2) The females must be nulliparous and nonpregnant.
(3) In acute toxicity tests with animals of a higher order than
those mentioned above, the use of smaller numbers should be considered.
(D) Assignment of animals. Each animal must be assigned a unique
identification number. A system to randomly assign animals to test
groups and control groups is required.
(E) Housing. Animals should be housed in individual cages.
(1) The temperature of the experimental animal rooms should be at 22
3 [deg]C for rodents, 20 3
[deg]C for rabbits.
(2) The relative humidity of the experimental animal rooms should be
30 to 70%.
(3) Where lighting is artificial, the sequence should be 12-hours
light/12-hours dark.
(4) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water.
(iii) Dose levels and dose selection. (A) Three dose levels must be
used and spaced appropriately to produce test groups with a range of
toxic effects and mortality rates. The data must be sufficient to
produce a dose-response curve and permit an acceptable estimation of the
median lethal dose. Range finding studies using single animals may help
to estimate the positioning of the dose groups so that no more than
three dose levels will be necessary.
(B) Limit test. This test is described in paragraph (d)(2)(ii) of
this section.
(C) Vehicle. Solids should be pulverized when possible. The test
substance
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should be moistened sufficiently with water or, where necessary, a
suitable vehicle to ensure good contact with skin. If a vehicle or
diluent is needed, it should not elicit toxic effects itself nor
substantially alter the chemical or toxicological properties of the test
substance. In addition, the influence of the vehicle on penetration of
skin by the test substance should be taken into account. It is
recommended that wherever possible the use of an aqueous solution be
considered first, followed by consideration of a solution in oil (e.g.,
corn oil), and then by consideration of possible solution in other
vehicles. For nonaqueous vehicles the toxic characteristics of the
vehicle should be known, and if not known should be determined before
the test. Acceptable alternative vehicles include gum arabic, ethanol
and water, carboxymethyl cellulose, glycerol, propylene glycol, PEG
vegetable oil, and mineral oil as long as the vehicle is not irritating
and the inability to use water or saline is justified in the report.
(iv) Exposure and exposure duration. The test substance must be
administered over a period of 24 hours.
(v) Preparation of animal skin. Fur must be clipped from the dorsal
area of the trunk of the test animals. Shaving may be employed, but it
should be carried out at least 24 hours before dosing. Care must be
taken to avoid abrading the skin, which would alter its permeability.
(vi) Application of test substance. (A) The test substance must be
applied uniformly over a shaved or clipped area which is approximately
10% of the body surface area. The area starting at the scapulae
(shoulders) to the wing of the ileum (hip bone) and half way down the
flank on each side of the animal should be shaved or clipped. Liquid
test materials should be undiluted if possible. With highly toxic
substances, the surface area covered may be less, but as much of the
area as possible should be covered with as thin and uniform a film as
practical. The test material is not removed until 24 hours after
application. In the case where less than 10% of the surface area is
covered an approximation of the exposed areas should be determined.
(B) The test substance must be held in contact with the skin with a
porous gauze dressing (<8 ply) and nonirritating tape throughout a 24-
hour exposure period. The test site must be further covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance. Restrainers
may be used to prevent the ingestion of the test substance, but complete
immobilization is not a recommended method. Although a semiocclusive
dressing is preferred, an occlusive dressing will also be acceptable.
(C) At the end of the exposure period, residual test substance
should be removed where practicable using water or an appropriate
solvent.
(vii) Observation period. Although 14 days is recommended as a
minimum observation period, the duration of observation should not be
fixed rigidly. It should be determined by the toxic reactions, rate of
onset, and length of recovery period, and may thus be extended when
considered necessary. The time at which signs of toxicity appear, their
duration, and the time to death are important, especially if there is a
tendency for deaths to be delayed.
(viii) Observation of animals. (A) A careful clinical examination
must be made at least once each day.
(B) Additional observations must be made daily, especially in the
early days of the study. 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 of weak or moribund animals).
(C) Observations must 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,
respiratory and circulatory effects, autonomic effects such as
salivation, central nervous system effects, including tremors and
convulsions, changes in the level of activity, gait and posture,
reactivity to handling or sensory stimuli, altered strength, and
stereotypies or bizarre behavior (e.g., self-mutilation, walking
backwards).
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(D) Individual weights of animals must be determined shortly before
the test substance is administered, weekly thereafter, and at death.
Changes in weights should be calculated and recorded when survival
exceeds one day.
(E) The time of death should be recorded as precisely as possible.
(ix) Gross pathology. (A) At the end of the test, surviving animals
must be weighed and sacrificed.
(B) A gross necropsy must be performed on all animals under test.
All gross pathology changes should be recorded.
(C) If necropsy cannot be performed immediately after a dead animal
is discovered, the animal should be refrigerated (not frozen) at
temperatures low enough to minimize autolysis. Necropsies should be
performed as soon as practicable, normally within a day or two.
(x) Additional evaluations. Microscopic examination of organs
showing evidence of gross pathology in animals surviving 24 hours or
more should also be considered because it may yield useful information.
(xi) Data and reporting--(A) Treatment of results. Data must be
summarized in tabular form, showing for each test group the number of
animals at the start of the test, body weights, time of death of
individual animals at different dose levels, number of animals
displaying other signs of toxicity, description of toxic effects and
necropsy findings. Any methods used for calculation of the
LD50 or any other parameters should be specified and
referenced. Methods for parameter estimation are described in the
references listed in paragraphs (f)(1), (f)(2), and (f)(3) of this
section.
(B) Evaluation of results. An evaluation should include the
relationship, if any, between exposure of the animals to the test
substance and the incidence and severity of all abnormalities, including
behavioral and clinical abnormalities, gross lesions, body weight
changes, effects on mortality, and any other toxic effects. The
LD50 value should always be considered in conjunction with
the observed toxic effects and any necropsy findings. The
LD50 value is a relatively coarse measurement, useful only as
a reference value for classification and labeling purposes, and for an
expression of the lethal potential of the test substance by the dermal
route. Reference should always be made to the experimental animal
species in which the LD50 value was obtained.
(C) Test report. In addition to the reporting requirements specified
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J, the following specific information must be reported. The test report
must include:
(1) Species, strain, sex, and source of test animals.
(2) Method of randomization in assigning animals to test and control
groups.
(3) Rationale for selection of species, if other than that
recommended.
(4) Tabulation of individual and test group data by sex and dose
level (e.g., number of animals exposed, number of animals showing signs
of toxicity and number of animals that died or were sacrificed during
the test).
(i) Description of toxic effects, including their time of onset,
duration, reversibility, and relationship to dose.
(ii) Body weights.
(iii) Time of dosing and time of death after dosing.
(iv) Dose-response curves for mortality and other toxic effects
(when permitted by the method of determination).
(v) Gross pathology findings.
(vi) Histopathology findings and any additional clinical chemistry
evaluations, if performed.
(5) Description of any pre-test conditioning, including diet,
quarantine and treatment for disease.
(6) Description of caging conditions including: Number (or change in
number) of animals per cage, bedding material, ambient temperature and
humidity, photoperiod, and identification of diet of test animals.
(7) Manufacturer, source, purity, and lot number of test substance.
(8) Relevant properties of substance tested including physical state
and pH (if applicable).
(9) Identification and composition of any vehicles (e.g., diluents,
suspending agents, and emulsifiers) or other materials used in
administering the test substance.
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(10) A list of references cited in the body of the report.
References to any published literature used in developing the test
protocol, performing the testing, making and interpreting observations,
and compiling and evaluating the results.
(f) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available for inspection at the TSCA Nonconfidential
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Chanter, D.O. and Heywood, R., The LD50 Test: Some
Considerations of Precision, Toxicology Letters 10:303-307 (1982).
(2) Finney, D.J. Chapter 3--Estimation of the median effective dose
and Chapter 4-Maximum likelihood estimation, Probit Analysis, 3rd ed.
Cambridge, London (1971).
(3) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives
of Toxicology 56:215-218 (1985).
(4) Organization for Economic Cooperation and Development. OECD
Guideline for the Testing of Chemicals. OECD Guideline 425: Acute Oral
Toxicity: Up-and-Down Procedure. Adopted: September 21, 1998.
(5) Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Guideline 420: Acute Oral
Toxicity--Fixed Dose Method. Adopted: July 17, 1992.
(6) Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Guideline 423: Acute Oral
Toxicity--Acute Toxic Class Method. Adopted: March 22, 1996
(7) Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal
Toxicity. Adopted: February 24, 1987.
[65 FR 78774, Dec. 15, 2000]