ASTM E1163-10(2016)
(Test Method)Standard Test Method for Estimating Acute Oral Toxicity in Rats
Standard Test Method for Estimating Acute Oral Toxicity in Rats
SIGNIFICANCE AND USE
5.1 This test method is of principal value in minimizing the number of animals required to estimate the acute oral toxicity (LD50). It also incorporates measures of variance (95 % CI) and a slope from which to make relative toxicity comparisons.
5.2 This test method is inappropriate for materials typically producing death two or more days after administration of the test compound unless the observation time between dosages is increased. This test method can be successfully applied, however, for materials producing only an occasional death two or more days after administration.
5.3 The LD50 is valuable as a measure of the relative acute toxicity of a material and can be used to make an estimate of potential hazard to humans when pesticides, other chemicals, or mixtures are ingested.
5.4 This test method allows for observation of signs of toxicity in addition to mortality. This information can be useful in planning additional toxicity testing.
SCOPE
1.1 This test method determines the lethality (LD50 value, slope and 95 % confidence interval (CI)) and signs of acute toxicity from a material using a limited number of rats. The technique used in this test method is referred to as the “Stagewise, Adaptive Dose Method.”2 This test method is an alternative to the classical LD50 test and is applicable to both liquids and solids.
1.2 This test method is not recommended for test materials which typically produce deaths beyond two days postdosing.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E1163 − 10 (Reapproved 2016)
Standard Test Method for
Estimating Acute Oral Toxicity in Rats
This standard is issued under the fixed designation E1163; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.3 LD50—thestatisticallyderivedestimateofthedoseof
atestsubstancethatwouldbeexpectedtocause50%mortality
1.1 This test method determines the lethality (LD50 value,
to the test population under the specified test conditions.
slope and 95% confidence interval (CI)) and signs of acute
3.1.4 moribund—at the point of death or extinction.
toxicity from a material using a limited number of rats. The
3.1.5 pharmacotoxic—grossphysiologicalsignsinresponse
technique used in this test method is referred to as the
2 to a toxic material.
“Stagewise, Adaptive Dose Method.” This test method is an
3.1.6 signs of toxicity—objective, observable evidence of
alternative to the classical LD50 test and is applicable to both
toxicity.
liquids and solids.
3.1.7 suspension—a mixture in which very small particles
1.2 This test method is not recommended for test materials
remain suspended without dissolving.
which typically produce deaths beyond two days postdosing.
3.1.8 toxicity—poisonous quality.
1.3 This standard does not purport to address all of the
4. Summary of Test Method
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4.1 Three to five different doses of the target compound are
priate safety and health practices and determine the applica-
selected such that the doses span the entire dose response
bility of regulatory limitations prior to use.
curve, with separation between the doses to be equal log
intervals. One to two animals are given each dose as the first
2. Referenced Documents
stage of the study.After 24 to 48 h, the responses to each dose
are observed and used in determining the doses and animal
2.1 ASTM Standards:
numbers in the next stage of dosing.
E609Terminology Relating to Pesticides
IEEE/ASTM SI10Standard for Use of the International
4.2 The second and subsequent stages have one to four
System of Units (SI) (the Modernized Metric System)
doses with one to three animals at each dose. Doses for
subsequent stages are selected based on the estimates of the
3. Terminology
dose response distribution parameters and the uncertainties of
theseestimates.Thedoseresponsecurveanditsparametersare
3.1 Definitions:
updated after each stage and dosing will stop when the 95 %
3.1.1 delayed death—an animal which does not die or
confidence interval for the LD50 satisfies the following stop-
appear moribund within 24 h but dies later during the obser-
ping rule: (upper 95 % CI – lower 95 % CI)/ (2 × LD50) < =
vation period.
0.40).
3.1.2 gavage—forcedoraldosing,asbyatubethatispassed
down the throat to the stomach. 4.3 The slope, LD50 and its 95 % confidence interval are
calculated using the methods Feder. No more than the use of
30 animals is recommended and shall constitute an additional
stopping rule.
This test method is under the jurisdiction of ASTM Committee E50 on
Environmental Assessment, Risk Management and Corrective Action and is the
5. Significance and Use
direct responsibility of Subcommittee E50.47 on Biological Effects and Environ-
mental Fate.
5.1 This test method is of principal value in minimizing the
Current edition approved Feb. 1, 2016. Published March 2016. Originally
number of animals required to estimate the acute oral toxicity
approved in 1987. Last previous edition approved in 2010 as E1163–10. DOI:
10.1520/E1163-10R16.
(LD50). It also incorporates measures of variance (95 % CI)
Feder,P.I.,StatisticalDesignConsiderationsforStagewise,stagewise,adaptive
and a slope from which to make relative toxicity comparisons.
doseallocationindoseresponsivestudies.In:Peace,KarlE.,ed.Biopharmaceutical
sequential statistical applications. New York: Marcel Dekker 1992.
5.2 This test method is inappropriate for materials typically
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
producing death two or more days after administration of the
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
test compound unless the observation time between dosages is
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. increased. This test method can be successfully applied,
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1163 − 10 (2016)
however,formaterialsproducingonlyanoccasionaldeathtwo 10. Procedure
or more days after administration.
10.1 Deprive the animals of food for 12 to 18 h before
5.3 The LD50 is valuable as a measure of the relative acute administering the test substance.
toxicity of a material and can be used to make an estimate of
10.2 Weight of each rat and calculate the dose according to
potential hazard to humans when pesticides, other chemicals,
thisbodyweighttogivethespecifiedquantityoftestsubstance
or mixtures are ingested.
per unit of body weight.
5.4 This test method allows for observation of signs of
10.3 Record all information necessary to document animal
toxicityinadditiontomortality.Thisinformationcanbeuseful
weights and volume of test substance administered to each
in planning additional toxicity testing.
animal.
10.4 Dose four to six animals in the first stage each at a
6. Apparatus
differentdosespanningtheestimateddoseresponsecurve(0%
6.1 Syringe, and an oral dosing needle or rubberized cath-
lethality to 100 % lethality). Gavage animals using an oral
eter to gavage the test compound is required.
dosing needle or rubberized tubing. Observe each animal for a
minimum of 24 h. Use the methods of Feder et al (1992) to
7. Test Animals
estimate the dose response curve and its parameters. Check to
7.1 Albino female rats weighing 190 to 300 g prefasted are
see if the stopping rule (defined in 4.2) is met. If not, then
used. A non in-bred rat such as the Sprague-Dawley strain is
proceed to the second stage of dosing. Return the animals to
generally preferred. Female rats are preferred because histori-
either ad libitum or 2 to 3-h feeding immediately after dosing.
cal data indicate that females in most instances have lower
Return the animal to either ad libitum or 2 to 3-h feeding
LD50 values than males.
immediately after dosing.
7.2 An additional test may be conducted with male rats, but
10.5 Observe the animals for mortality and pharmatoxic
it is not necessary, unless it is suspected that the substance is
signs periodically for the first 4 h after dosing (at least once
more toxic to males than females.
during the first 30 min) and daily thereafter for a total of seven
days.
8. Pretest Conditioning
10.6 Pharmatoxicsignsmostfrequentlyseenareasfollows:
8.1 Examine each test animal on arrival for overt signs of
respiratory rate increase or decrease, hypoac
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1163 − 10 E1163 − 10 (Reapproved 2016)
Standard Test Method for
Estimating Acute Oral Toxicity in Rats
This standard is issued under the fixed designation E1163; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method determines the lethality (LD50 value, slope and 95%95 % confidence interval (CI)) and signs of acute
toxicity from a material using a limited number of rats. The technique used in this test method is referred to as the “Stagewise,
Adaptive Dose Method.” This test method is an alternative to the classical LD50 test and is applicable to both liquids and solids.
1.2 This test method is not recommended for test materials which typically produce deaths beyond 2two days postdosing.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2.1 ASTM Standards:
E609 Terminology Relating to Pesticides
IEEE/ASTM SI 10 Standard for Use of the International System of Units (SI) (the Modernized Metric System)
3. Terminology
3.1 Definitions:
3.1.1 delayed death—an animal which does not die or appear moribund within 24 h but dies later during the observation period.
3.1.2 gavage—forced oral dosing, as by a tube that is passed down the throat to the stomach.
3.1.3 LD50—the statistically derived estimate of the dose of a test substance that would be expected to cause 50 % mortality
to the test population under the specified test conditions.
3.1.4 moribund—at the point of death or extinction.
3.1.5 pharmacotoxic—gross physiological signs in response to a toxic material.
3.1.6 signs of toxicity—objective, observable evidence of toxicity.
3.1.7 suspension—a mixture in which very small particles remain suspended without dissolving.
3.1.8 toxicity—poisonous quality.
4. Summary of Test Method
4.1 Three to five different doses of the target compound are selected such that the doses span the entire dose response curve,
with separation between the doses to be equal log intervals. One to two animals are given each dose as the first stage of the study.
After 24 to 48 h, the responses to each dose are observed and used in determining the doses and animal numbers in the next stage
of dosing.
4.2 The second and subsequent stages have one to four doses with one to three animals at each dose. Doses for subsequent
stages are selected based on the estimates of the dose response distribution parameters and the uncertainties of these estimates. The
This test method is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Action and is the direct
responsibility of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Nov. 1, 2010Feb. 1, 2016. Published June 2011March 2016. Originally approved in 1987. Last previous edition approved in 20022010 as
E1163 – 98E1163 – 10.(2002). DOI: 10.1520/E1163-10.10.1520/E1163-10R16.
Feder, P.I., Statistical Design Considerations for Stagewise, stagewise, adaptive dose allocation in dose responsive studies. In: Peace, Karl E., ed. Biopharmaceutical
sequential statistical applications. New York: Marcel Dekker 1992.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1163 − 10 (2016)
dose response curve and its parameters are updated after each stage and dosing will stop when the 95 % confidence interval for
the LD50 satisfies the following stopping rule: (upper 95 % CI – lower 95 % CI)/ (2 × LD50) < = 0.40).
4.3 The slope, LD50 and its 95 % confidence interval are calculated using the methods Feder. No more than the use of 30
animals is recommended and shall constitute an additional stopping rule.
5. Significance and Use
5.1 This test method is of principal value in minimizing the number of animals required to estimate the acute oral toxicity
(LD50). It also incorporates measures of variance (95 % CI) and a slope from which to make relative toxicity comparisons.
5.2 This test method is inappropriate for materials typically producing death 2two or more days after administration of the test
compound unless the observation time between dosages is increased. This test method can be successfully applied, however, for
materials producing only an occasional death 2two or more days after administration.
5.3 The LD50 is valuable as a measure of the relative acute toxicity of a material and can be used to make an estimate of
potential hazard to humans when pesticides, other chemicals, or mixtures are ingested.
5.4 This test method allows for observation of signs of toxicity in addition to mortality. This information can be useful in
planning additional toxicity testing.
6. Apparatus
6.1 Syringe, and an oral dosing needle or rubberized catheter to gavage the test compound is required.
7. Test Animals
7.1 Albino female rats weighing 190 to 300 g prefasted are used. A non in-bred rat such as the Sprague-Dawley strain is
generally preferred. Female rats are preferred because historical data indicate that females in most instances have lower LD50
values than males.
7.2 An additional test may be conducted with male rats, but it is not necessary, unless it is suspected that the substance is more
toxic to males than females.
8. Pretest Conditioning
8.1 Examine each test animal on arrival for overt signs of disease, and condition to the environment for a minimum of 7seven
days. Select animals that have not been used for any other tests.
8.2 Maintain animals during pretest and test periods in accordance with accepted laboratory practices for the care and handling
of test animals.
8.3 Identify each animal with an ear tag or other suitable means.
8.4 During acclimation, observe the animals for adverse health effects. Eliminate any animal(s) demonstrating signs of
spontaneous disease prior to the start of the study. Use only animals judged to be healthy.
8.5 The animals are housed individually. Rat chow or the equivalent and water are to be available ad libitum after dosing.
9. Sample Preparation
9.1 Because of the great variety of physical characteristics and formulations of chemicals and pesticides, it is not possible to
stipulate how the test material should be prepared. The only criterion that can be specified is that the material must be in liquid
form, that is liquid, solution, suspension, or emulsion, suitable for administration by gavage.
9.2 The test material shall be at the same temperature as that of the room in which the test is conducted at the time of
administration to t
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