Pesticide Assessment Guidelines, Subdivision F
Revised Policy for Acute Toxicity Testing
(U.S.) Environmental Protection Agency, Washington, DC
22 Sep 88
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J
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•nd Subtitle
;ticide Assessment Guidelines, Subdivision F, Revised
licy for Acute -Toxicity Testing
7. AutlwxU)
EPA/HEP
9. Performlnf Orfanlratloo Name and Address
U.S. ENVIRONMENTAL PROTECTION AGENCY
OPP/HED (H7509C)
401 M St., S.W.
WASHINGTON, D.C. 20460
12.' Sponsoring Organization "*me and Address
Same as #9
IS. Supplementary Notes
For more information, refer to Subdivision F: Series 81-1, 81-2 and 81-3
Addendum 6 PB89-124077
(. Report Dale
9/22/88
(. Performing OrcanUetlon Rept. No.
540/09-90-072
10. Prolect/Task/Work Unit No.
11. Contract(C) or Cr«nl(G) No.
(G)
13. Type of Report & Period Covered
16. Abstract (Limit: ?00 words)
In response to the need to improve scientific and regulatory decisions and to
provide additional guidance for evaluating the acute toxicity of chemical exposures
under the Federal Insecticide, Fungicide and Rodenticide Act and the Toxic Substances
£t>ntrol Act. This action builds upon a previous revision of the acute toxicity
esting strategy tc reduce the use of experimental animals whii'e providing adequate
formation about chemical safety.
The Environmental Protection Agency is disseminating this notice to industry,
governmental bodies., scientific societies, animal welfare groups and interested
parties to apprise them of our new position. The Agency's acute toxicity
testing guidelines are being revised to reflect the positions articulated in this
policy.
17. Document Analysis a. Descriptors
b. Id«ntifi«rs/Op*n-Cnd«d
c. COSAtl ricld/G'Oun
(•liability Statemen:
nciassifled
REPRODUCED BY
U.S. DEPARTMENT OF COMMERCE
NATIONAL TECHNICAL INFORMATION SERVICE
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19. Security Class (Tnis Report)
Unclassified _
70. Security Class (This face)
Unclassified
21. No. of Pagrs
22. Price
(See ANSI-Z.39.18>
See fnefrveffofta on Reverse
OPTIONAL FORM 272 (4-77)
(formerly NTIS-31)
Department of Commerce
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545T; 09-90-072
\ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
I WASHINGTON. DC. 20460
y
orrici e*
ritTICIDU AND TOXIC iU««TA~CI
22 1988
Re: Revised Policy for Acute Toxicity Testing
**
Appended is a revised policy for evaluating the acute
toxicity of chemical exposures under the Federal Insecticide.
Fungicide and Rodeziticide Act and the Toxic Substances Control
Act. This action builds upon a previous revision of the acute
tox?city testing strategy to reduce the use of experimental
animals while providing adequate information about chemical
safety.
The Environmental Protection Agency is disseminating this
notice to industry, governmental bodies, scientific societies,
animal welfare groups and interested parties to apprise them of
our new position. The Agency's acute toxicity testing guidelines
are being revised to reflect the positions, articulated in this
policy.
Victor J. Aimm
Acting i^sistant Administrator
for Pesticides
and Toxic Substances
Enclosure
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Alternative Methodology for Acute Toxicity Testing
The Environmental Protection Agency announces a revision to
its approach to acute toxicity testing in fulfillment of actions
under the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA) and the Toxic Substances Control Act (TSCA). This
revision reflects the Agency's concern about animal welfare and
its continued efforts to reduce the impacts on animals of EPA's
testing requirements. While maintaining the tiered approach
adopted in 1984, the Agency now recommends (when appropriate) the
use of abbreviated test methods and consideration of using only
one sex, as a means of reducing the numbers of animals in
deriving important information on acute toxicity.
Background
EPA considers the evaluation of toxicity following short-
term exposure to a chemical (i.e., acute toxicity) to be a
limited but integral step in the assessment of the toxic
potential of a chemical substance under the regulatory framework
of its pesticide and toxic substances programs. The Agency also
supports measures dedicated to reduce the use of animals in
toxicity testing and conducts research on test methods which can
lead to further reduction or elimination of animal usage and
suffering. Through the careful selection of test methodology and
maximization of the data obtained from acute studies, EPA strives
to achieve a balance between the welfare of animals and the need
to utilize animals in evaluating chemical safety.
The approach to acute toxicity testing previously given
in EPA's Test Guidelines (U.S. Env. Prot. Agency, 1978; 1979)
emphasized the determination of the median lethal dose (LD50)
with a 95% confidence interval. A 1984 update of the guidelines,
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published in 1985 (U.S. Env. Prot. Agency, 1985) stated that the
Agency discouraged the uses of the "classical" LD50 test
employing large numbers of animals for determination of lethality
only. Instead, the Agency emphasized the use of a tiered
approach to obtain acute toxicit.' data which reduced the number
of animals used, but maximized the amount of relevant
information that could be obtained from such testing. That
approach included the following:
a. Using Data From Structurally Related Chemicals, The Agency
encourages the review of existing acute toxicity information
on chemical substances that are structurally related to the
agent under investigation. Using this approach, one may be
able to compile enough information from these surrogate
chemicals to make preliminary safety evaluations that
reduce the need for further animal testing or which
indicate the type of testing to be pursued.
b. "Limit" Test. When information on structural analogs is
inadequate, one should consider the "limit" test. The
relative toxicity of a chemical is determined by
professional judgement; for chemicals judged to be
relatively non-toxic, a single group of animals is given a
large dose of the agent. If no lethality is demonstrated,
no further testing for this information IB pursued.
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c. Multifaceted Testing. A three-dose multiple endpoint
evaluation may be important for those substances judged to
be relatively toxic or which demonstrate lethality in the
limit test. Using this procedure, animals are evaluated as
to the onset, duration, intensity, and reversibility of
behavioral effects, body weight changes and lethality; all
animals are submitted to gross necropsy. Histopatholcay and
certain follow-up studies may be warranted where t^re are
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gross indications of target organ toxicity.
PresentRevision.
EPA has reevaluated its data needs on acute toxicity and
continues to espouse the tiered approach that was developed in
the 1984 update. Thus, the first consideration for a chemical
for which there is no acute toxicity data, should be a review of
structurally related compounds, followed by the limit test when
appropriate. In those cases where testing beyond the limit test
is indicated, consideration should be given to well-designed
abbreviated test schemes which employ minimal numbers of animals,
«s discussed below. In most cases, it is expected that these
tests can be structured to give enough information on acute
toxicity to obviate the need for further acute studies (e.g., the
three-dose multi-faceted testing approach). We continue to
stress the need for collecting information on behavioral
effects, gross pathology and lethality (as developed in "c"
above).
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While more complete animal testing may be necessary in some
cases (based on scientific evidence from the abbreviated test,
e.g., delayed toxicity, unusual central nervous system effects,
irreversible effects), the Agency generally supports limiting
such tests to those using the lowest feasible number of animals.
Several abbreviated methods to investigate acute toxicity
have been developed over the years. Some of them have rather
extensive data bases and have been validated against more
traditional test methods which estimate median lethal dose.
Their merit lies in the fact that they allow for the evaluation
of the full spectrum of acute responses; numerical calculations
can be made; and fewer animals may be employed in the generation
of the information than with most other approaches. For some
methods, statistical calculations are simple or are aided by
tables.
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5
EPA has investigated four methodologies that might be used.
These include (1) the approximate lethal dose method1 of
Deichmann and Le Blanc (1943); (2) the moving averages method2 of
Thompson (1947); (3) the up-and-down method3 of Dixon and Mood
(1948) and Dixon (1965); and (4) the cumulant method of Reed-
Muench4 (1938) .
The methods vary as to the assumptions that are made, the
number of groups of animals and number of animals per group.
Toxicologists Should be familiar with these differences before
employing a given method. For instance, the up-and-down method
is especially difficult to apply when chemicals induce delayed
toxic effects. Therefore, other methodologies may be more
appropriate. When an alternative method for acute toxicity
testing is selected, a rationale for such a selection should
accompany the submission. The Agency solicits discussions with
data generators on still other methods that may be employed.
1The approximate lethal dose method was further refined by
Deichmann and Mergard (1948) ; these authors performed eighty-
seven determinations (calculated by the methods of Behrens
(1929) and Bliss (1938)). The approximate lethal dose method was
also used by Kennedy et al. (1986).
2The moving averages method was refined by Weil (1952, 1983)
and Gad and Weil (1982), and was used by Smythe and Carpenter
(1944, 1948) and Smythe et al. (1949, 1951, 1954, 1962).
up-and-down method was recently used and refined by
Bruce (1985, 1987) (calculated by the method of Bliss (1938)).
The up-and-down method was also used by Brownlee et al. (1953),
Oixon and Hassey (1957), Klassen ana Plaa (1967) and Hsi (1969).
4The Department of Defense has had considerable experience
using the Reed-Muench method with a large number of chemicals (F.
Vocci, personal communication); it has also been used by Lorenz
and Bogel (1973), Bhan (1974), Aubert and Amdral (1979), and
Thakur and Fezio (1981).
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The Agency emphasizes that parallel assays on ir.ale ar.J
female animals to determine an approximate estimate of acute
toxicity need not be routinely determined, since male and female
animals of the same strain generally show only slight and
insignificant differences in susceptibility to toxic agents.
However, for some chemicals, one sex may be somewhat more
sensitive than-*.he other (Muller and Kley (1982); Schutz and
Fuchs (1982); (Bruce (1985)). Cassarett and Doull (1980)
indicate that the class of compound is important in specific sex
differences. De Pass et al. (1984) shoved that for 91 chemicals
tested for oral toxicity in rats, females were slightly more
sensitive than males (p<.001). Muller and Kley (1982) performed
152 parallel studies on male and female animals for which 129
showed no significant differences. However, when statistically
significant differences were observed (23 compounds), 17 were
more toxic to females. Therefore, consideration should be given
to limiting studies to the more sensitive sex. Previous history
on the class of chemical being evaluated would be helpful in
making this determination. For confirmation, a few animals of
the other sex should also be tested.
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7
In summary, EPA has modified its approach to acute toxicity
testing, recognizing that appropriate information for safety
evaluation can be developed using fewer animals than had been
recommended in the past. We strongly urge industry to use these
abbreviated test methodologies, whenever appropriate, as
replacements for the three-dose multifaceted method EPA
previously had recommended. Four such methodologies which might
be used have been identified; other methods may also be employed,
if adequate rationale can be provided. It IB expected that
studies will still include behavioral observations, gross
necropsy and ancillary observations, as before.
EPA urges industry to begin submitting data obtained with
alternate methods which use fewer animals on a routine basis;
the Agency is planning to revise its testing guidelines to
incorporate the above guidance. We plan to accept only newly
generated industry data that conforms with our revised guidance
unless an adequate rationale (e.g., data generated in accordance
with regulatory requirements other than those of EPA) accompanies
the submission; data without a rationale may be returned to the
submitter.
The Agency encourages the public to comment on this
position and provide information on still other alternate
methodologies which have progressed to a stage of validation
which would be acceptable to the scientific community.
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References
Aubert M.F.A. and Ajndral L. (1979). Potency Testing of
Veterinary Vaccines Containing Inactivated Virus. Coir.p. Inununol
Microbiol. Infect. Dis., 1, 341-349.
Behrens B. (1929). The Assay of Digitalis on Frogs. Arch.
Exptl. Path. Pharmakol., 140. 237-256.
Bhan A.K. (1974). Computing LD50 More Efficiently and Accurately
- A Modification of Reed-Muench Method. Indian Soc. Nucl.
Agricul. Biol., l, 5-6.
Bliss C.I. (1938). Determination of the Small Dosage Mortality
Curve from SmaM Numbers. Quart. J. Year Book Pharro., 11. 192-
216.
Brownlee K.A., Hodges J.L. and Rosenblatt M. (1953). The Up-and-
Down Method With Small Samples. Amer. Statist. Assoc. J., 48.
262-277.
Bruce R.D. (1985). An Up-and-Dovn Procedure for Acute Toxicity
Testing. Fundam. Appl. Toxicol., 5_, 151-157.
Bruce R.D. (1987). A Confirmatory Study of the Up-and-Dovn
Method for Acute Oral Toxicity Testing. Fundam. Appl. Toxicol.,
8, 97-100.
Casarett L.J. and Doull J. (1980). Factors Influencing
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Kacmillan Publishing Co., Inc.
Deichmann W.B. and LeBlanc T.L. (1943). Determination of the
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Deichmann W.B. and Mergard E.G. (1948). Comparative Evaluation
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De Pass L.R., Myers R.C., Weaver E.V. and Weil C.S. (1984). An
Assessment of the Importance of Number of Dosage Levels, Number
of Animals per Dosage Level, Sex and Method of LD50 and Slope
Calculation in Acute Toxicity Studies. In: Acute Toxicity
Testing; Alternative Approaches. A.M. Goldberg, «d. (Alternative
Methods in Toxicology, Vol. 2), 142-153. New York: Mary A.
Liebert, Inc., Publisher.
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9
References (contd,)
Dixon W.J. (1965). The Up-and-Dovn Method for Small Samples.
Arner. Statist. Assoc. J., 60, 967-978.
Dixon W.J. and Massey F.J. Jr. (1957). Sensitivity Experiments,
In: Introduction to Statistical Analysis. 318-327. New York:
McGraw-Hill.
Dixon W.J. and Mood A.M. (1948). A Method for Obtaining and
Analyzing Sensitivity Data. Ainer. Statist. Assoc. J., 4 3. 109-
126.
Gad S.C. and Weil C.S. (1982). Statistics for Toxicologists.
In: Methods in Toxicology. A.W. Hays, ed., 273-309. New York:
Raven Press. ^
Hsi, B.P. (1969). The Multiple Sample Up-and-Down Method in
Bioassay. Amer. Statist. Assoc. J., 64. 147-162.
Kennedy, G.L. Jr., Ferenz, R.L. and Burgess, B.A. (1986).
Estimation of Acute Oral Toxicity in Rats by Determination of the
Approximate Lethal Dose Rather Than the LD50. J. Appl. Toxicol.
6, 145-148.
Klassen, C.D. and Plaa, G.L. (1967). Relative Effects of Various
Chlorinated Hydrocarbons on Liver and Kidney Function in Dogs.
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Lorenz R.J. and Bogel K. (1973). Methods of Calculation. Method
of Reed and Muench. Laboratory Techniques in Rabies. WHO
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Muller H. and Kley H.P. (1982). Retrospective Study on the
Reliability of an "Approximate LD50"5 Determined with a Small
Number of Animals. Arch. Toxicol., 51, 189-196.
Reed L.J. and Muench H. (1938). A Simple Method for Estimating
Fifty Percent Endpoints. Amer. J. Indust. Hyg. Toxicol., 30,
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Schutz E. and Fuchs H. (1?B2). A New Approach to Minimizing the
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5 "Approximate LD50" in the title of this paper is placed ir,
quotation marks eo as not to confuse it with the method of
Deichman and Le Blanc (1943).
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10
References (contd.)
Snythe H.F. Jr. and Carpenter C.P. (1948). Further Experience
with the Range-Finding Test in the Industrial Toxicology
Laboratory. J. Indust. Hyg. Toxicol., 30. 63.
Smythe H.F. Jr., Carpenter C.P. and Weil C.S. (1949). Range-
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60.
Sirythe H.F. Jr., Carpenter C.P. and Weil C.S. (1951). Range-
Finding Toxicity Data: List IV. AKA Arch. Indust. Hyg. Occ.
Med., 4; 119.
Smythe H.F. Jr., Carpenter C.P., Weil C.S. and Pozzani U.C.
(1954). RangeeFinding Toxicity Data: List V. AMA Arch. Indust.
Hyg. Occ. Med. 10, 61.
Smythe H.F. Jr., Carpenter C.P., Weil C.S., Pozzani U.C., and
Striegel J.A. (1962). Range-Finding Toxicity Data: List VI.
Amer. Indust. Hyg. Assoc. J., 23. 95.
Thakur A.K. and Fezio W.L. (1981). A Computer Program for
Estimating LD50 and It's Confidence Limits Using Modified
Behrens-Reed-Kuench Cumulant Method. Drug Chem. Toxicol., 4.,
297-305.
Thompson, W.R. (1947). Use of Moving Averages and Interpolation
to Estimate Median Effective Dose. Bacteriol. Rev. 11. 115-145.
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Guidelines for Registering Pesticides in the U.S. Hazard
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37345; 37351-37356. (August 22).
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Effects Test Standards for Toxic Substances Control Act Test
Rules and Proposed Good Laboratory Practice Standards for Health
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U.S. Environmental Protection Agency. (1985). Health Effects
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Vocci. F. (1988). Personal Communication (Unpublished Data).
Weil C.S. (1952). Tables for Convenient Calculation of Median
Effective Dose (LD50 or ED50) *nd Instructions on Their Use.
Biometrics 8, 249-263.
Weil C.S. (1983). Economical LD50 and Slope Determinations.
Drug Chem. Toxicol. £, 595-603.
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