[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: 40CFR799.9110]
[Page 321-326]
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. 799.9110 TSCA acute oral toxicity.
Source: 62 FR 43824, Aug. 15, 1997, unless otherwise noted.
(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 oral toxicity is usually an initial step. It
provides information on health hazards likely to arise from short-term
exposure by the oral route. Data from an acute study may serve as a
basis for classification and labeling. It is traditionally a step in
establishing a dosage regimen in subchronic and other studies and may
provide initial information
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on the mode of toxic action of a substance. 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 870.1100 (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 oral toxicity is the adverse effects occurring within a short
period of time after oral administration of either 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 administered. Dose is expressed
as weight of test substance (milligrams, grams) 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) is a statistically derived
estimate of single dose of a substance that can be expected to cause
death in 50% of animals when administered by the oral route. The
LD50 value is expressed in terms of weight of test substance
per unit weight of test animal (milligrams per kilogram).
(d) Alternative approaches to the determination of acute toxicity.
(1) EPA will accept the following procedures to reduce the number of
animals used to evaluate acute effects of chemical exposure while
preserving its ability to make reasoned judgments about safety:
(i) Estimation of acute oral toxicity. When further study is
warranted, EPA generally supports limiting such tests to those using the
lowest number of animals feasible. EPA will accept three alternative
Organization for Economic Cooperation and Development (OECD) test
methods in place of the ``traditional'' acute oral toxicity test. The
three OECD alternatives are the following:
(A) The up and down procedure as described in OECD Guideline 425
referenced in paragraph (f)(4) of this section.
(B) The acute toxic class method as described in OECD Guideline 423
and referenced in paragraph (f)(6) of this section.
(C) The fixed dose method as described in OECD Guideline 420 and
referenced in paragraph (f)(5) of this section.
(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 per kilogram of body weight 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 oral toxicity is needed.
(Under current policy and regulations for pesticide products,
precautionary statements may still be required unless there are data to
indicate the LD50 is greater than 5,000 mg/kg.) If compound-
related mortality is produced in the limit test, further study may need
to be considered.
(2) [Reserved]
(e) Conventional acute toxicity test--(1) Principle of the test
method. The test substance is administered orally by gavage 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 rodent species but may be adapted
for studies in nonrodents. Animals showing severe
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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 described 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. Although several
mammalian test species may be used, the rat is the preferred species.
Commonly used laboratory strains must be employed. If another species is
used, the tester must provide justification and reasoning for its
selection.
(B) Age. Young adult rats between 8- and 12-weeks-old at the
beginning of dosing should be used. Rabbits should be at least 12 weeks
of age at study initiation. 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 rodents 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
rodents, the use of smaller numbers should be considered.
(D) Assignment of animals. Each animal must be assigned a unique
identification number. A system to assign animals to test groups and
control groups randomly is required.
(E) Housing. Animals may be group-caged by sex, but the number of
animals per cage must not interfere with clear observation of each
animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual
caging.
(1) The temperature of the experimental animal rooms should be at 22
3 [deg]C for rodents.
(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, spaced appropriately to produce test groups with a range of toxic
effects and mortality rates. The data collected must be sufficient to
produce a dose-response curve and permit an acceptable estimation of the
LD50. Range finding studies using single animals may help to
estimate the positioning of dose groups so that no more than three dose
levels will be necessary.
(B) Limit test. This test has been defined and described in
paragraph (d)(1)(ii) of this section.
(C) Vehicle. Where necessary, the test substance is dissolved or
suspended in a suitable vehicle. 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. 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. Toxic characteristics of nonaqueous vehicles should be known,
and, if not known, should be determined before the test.
(D) Volume. The maximum volume of liquid that can be administered at
one time depends on the size of the test
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animal. In rodents, the volume should not exceed 1 mL/100 g body weight,
except when an aqueous solution is used in which case 2 mL/100 g may be
administered. Either constant volume or constant concentration
administration is acceptable when dosing, provided the following
guidance is employed. When possible, the liquid test material should be
dosed neat. Otherwise, it may be diluted, using the highest
concentration possible, although volumes less than 0.5 mL per animal
would not be required. Lower dose volumes are acceptable if they can be
accurately administered. Solid materials should be suspended or
dissolved in the minimum amount of vehicle and dosed at the highest
concentration possible.
(iv) Exposure and exposure duration. (A) Animals must be fasted
prior to test substance administration. For the rat, feed should be
withheld overnight; for other rodents with higher metabolic rates a
shorter period of fasting is appropriate.
(B) The test substance must be administered in a single dose by
gavage, using a stomach tube or suitable intubation cannula.
(C) If a single dose is not possible, the dose may be given in
smaller fractions over a period not exceeding 24 hours. Where a dose is
administered in fractions, it may be necessary to provide the animals
with food and water, depending on the length of the dosing period.
(D) After the substance has been administered, feed may be withheld
for an additional 3-4 hours.
(v) 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.
(vi) 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).
(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 1 day.
(E) The time of death should be recorded as precisely as possible.
(vii) 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.
(viii) Additional evaluation. 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.
(ix) 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
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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
ingestion 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
shall 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 pretest 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.
(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
Guidelines for the Testing of Chemicals. OECD Guideline 425: Acute Oral
Toxicity: Up-and-Down Procedure, Approved: June 1998.
(5) Organization for Economic Cooperation and Development. OECD
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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 401: Acute Oral Toxicity,
Adopted: February 24, 1987.
[65 FR 78771, Dec. 15, 2000]