ASTM E1163-10(2019)

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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Jan-2019
• ICS: 65.100.99 - Other pesticides and agrochemicals
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Replaces: ASTM E1163-10(2016) - Standard Test Method for Estimating Acute Oral Toxicity in Rats
• Effective Date: 01-Feb-2019
• Refers: ASTM E609-16 - Standard Terminology Relating to Pesticides
• Effective Date: 15-Sep-2016
• Refers: ASTM E609-15a - Standard Terminology Relating to Pesticides
• Effective Date: 01-May-2015
• Refers: ASTM E609-15 - Standard Terminology Relating to Pesticides
• Effective Date: 01-Jan-2015
• Refers: ASTM E609-10 - Standard Terminology Relating to Pesticides
• Effective Date: 01-Mar-2010
• Refers: ASTM E609-05 - Standard Terminology Relating to Pesticides
• Effective Date: 01-Apr-2005
• Refers: ASTM E609-04 - Standard Terminology Relating to Pesticides
• Effective Date: 01-Nov-2004
• Refers: ASTM E609-03a - Standard Terminology Relating to Pesticides
• Effective Date: 01-Oct-2003
• Refers: ASTM E609-02a - Standard Terminology Relating to Pesticides
• Effective Date: 10-Oct-2002
• Refers: ASTM E609-03 - Standard Terminology Relating to Pesticides
• Effective Date: 10-Oct-2002
• Refers: ASTM E609-81(1997)e1 - Standard Terminology Relating to Pesticides
• Effective Date: 10-Apr-2001
• Refers: ASTM E609-00 - Standard Terminology Relating to Pesticides
• Effective Date: 10-Apr-2001
• Refers: ASTM E609-02 - Standard Terminology Relating to Pesticides
• Effective Date: 10-Apr-2001
• Referred By: ASTM E2552-23 - Standard Guide for Assessing the Environmental and Human Health Impacts of New Compounds for Military Use
• Effective Date: 01-Feb-2019

Overview

ASTM E1163-10(2019), Standard Test Method for Estimating Acute Oral Toxicity in Rats, provides a scientifically recognized approach for determining the acute oral toxicity (LD50 value, slope, and 95% confidence interval) of chemicals and mixtures using a minimal number of animals. This test method is particularly valued for its role in safety assessment and toxicological evaluation-important processes for manufacturers, regulatory bodies, and researchers handling pesticides, chemicals, or pharmaceuticals.

By employing the Stagewise, Adaptive Dose Method, this ASTM standard offers an alternative to classical LD50 testing and is suitable for both liquid and solid substances. Its design aims to maximize data collection while minimizing animal use, thus aligning with modern ethical standards and regulatory expectations in toxicological testing.

Key Topics

• Minimizing Animal Use: The procedure limits the number of rats used to a recommended maximum of 30, reducing animal testing while generating statistically robust toxicity data.
• LD50 Determination: Provides a method to calculate the median lethal dose (LD50), offering a direct measure of acute oral toxicity for risk evaluation.
• Adaptive Dosing Technique: Utilizes a staged approach, where dose levels and animal numbers are refined after each stage based on observed results, leading to greater accuracy and efficiency.
• Comprehensive Data Output: In addition to LD50 value, provides a slope (for toxicity comparison) and 95% confidence intervals, boosting the reliability of hazard assessments.
• Observational Insights: Records both mortality and observable signs of toxicity, contributing to broader safety profiling and supporting future studies.
• Guidance on Applicability: Advises against use for substances that typically cause delayed deaths beyond two days unless observation periods are extended.
• Reporting Requirements: Details essential report contents, including species, animal source, body weights, administered doses, mortality, pharmacotoxic signs, and necropsy findings.

Applications

ASTM E1163-10(2019) is commonly applied in:

• Regulatory Compliance: Supports registration and approval of chemicals, pesticides, and pharmaceuticals by providing standardized acute oral toxicity data for regulatory submissions.
• Product Safety Assessment: Utilized by manufacturers and toxicology laboratories to estimate potential human health hazards upon single oral exposure.
• Comparative Toxicology: Allows researchers to compare toxicity levels between different substances using the calculated LD50 and slope.
• Research and Development: Assists scientists in identifying toxicological profiles of new chemical entities and in planning additional in-depth toxicity studies based on observed effects.
• Risk Management: Supplies vital toxicological metrics needed for health and environmental risk assessments, influencing labeling, packaging, and safe handling procedures.

Related Standards

Several important standards are relevant to and often used alongside ASTM E1163-10(2019):

• ASTM E609 – Terminology Relating to Pesticides: Provides definitions critical for toxicological studies and regulatory clarity.
• IEEE/ASTM SI10 – Standard for Use of the International System of Units (SI): Offers guidance on consistent measurement units in laboratory settings.
• OECD Test Guidelines – Such as OECD TG 420, 423, and 425: Internationally recognized protocols for acute oral toxicity testing in animals.
• ASTM E729 – Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians: For testing toxicity in aquatic organisms.

---

Adopting ASTM E1163-10(2019) ensures scientifically valid and internationally recognized results in acute oral toxicity testing, facilitating regulatory acceptance and advancing animal welfare while ensuring the continued safety of chemicals and mixtures that enter the marketplace.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1163 − 10 (Reapproved 2019)
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 3.1.1 delayed death—an animal which does not die or
appear moribund within 24 h but dies later during the obser-
1.1 This test method determines the lethality (LD50 value,
vation period.
slope and 95% confidence interval (CI)) and signs of acute
3.1.2 gavage—forcedoraldosing,asbyatubethatispassed
toxicity from a material using a limited number of rats. The
down the throat to the stomach.
technique used in this test method is referred to as the
3.1.3 LD50—thestatisticallyderivedestimateofthedoseof
“Stagewise, Adaptive Dose Method.” This test method is an
atestsubstancethatwouldbeexpectedtocause50%mortality
alternative to the classical LD50 test and is applicable to both
to the test population under the specified test conditions.
liquids and solids.
3.1.4 moribund—at the point of death or extinction.
1.2 This test method is not recommended for test materials
3.1.5 pharmacotoxic—grossphysiologicalsignsinresponse
which typically produce deaths beyond two days postdosing.
to a toxic material.
1.3 This standard does not purport to address all of the
3.1.6 signs of toxicity—objective, observable evidence of
safety concerns, if any, associated with its use. It is the
toxicity.
responsibility of the user of this standard to establish appro-
3.1.7 suspension—a mixture in which very small particles
priate safety, health, and environmental practices and deter-
remain suspended without dissolving.
mine the applicability of regulatory limitations prior to use.
3.1.8 toxicity—poisonous quality.
1.4 This international standard was developed in accor-
dance with internationally recognized principles on standard-
4. Summary of Test Method
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom- 4.1 Three to five different doses of the target compound are
mendations issued by the World Trade Organization Technical
selected such that the doses span the entire dose response
Barriers to Trade (TBT) Committee. 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
3.1 Definitions:
theseestimates.Thedoseresponsecurveanditsparametersare
updated after each stage and dosing will stop when the 95 %
confidence interval for the LD50 satisfies the following stop-
This test method is under the jurisdiction of ASTM Committee E50 on
ping rule: (upper 95 % CI – lower 95 % CI)/ (2 × LD50) < =
Environmental Assessment, Risk Management and Corrective Action and is the
0.40).
direct responsibility of Subcommittee E50.47 on Biological Effects and Environ-
mental Fate.
4.3 The slope, LD50 and its 95 % confidence interval are
Current edition approved Feb. 1, 2019. Published February 2019. Originally
calculated using the methods Feder. No more than the use of
approved in 1987. Last previous edition approved in 2016 as E1163–10(2016).
DOI: 10.1520/E1163-10R19. 30 animals is recommended and shall constitute an additional
Feder,P.I.,StatisticalDesignConsiderationsforStagewise,stagewise,adaptive
stopping rule.
doseallocationindoseresponsivestudies.In:Peace,KarlE.,ed.Biopharmaceutical
sequential statistical applications. New York: Marcel Dekker 1992.
5. Significance and Use
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
5.1 This test method is of principal value in minimizing the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. number of animals required to estimate the acute oral toxicity
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1163 − 10 (2019)
(LD50). It also incorporates measures of variance (95 % CI) liquid form, that is liquid, solution, suspension, or emulsion,
and a slope from which to make relative toxicity comparisons. suitable for administration by gavage.
5.2 This test method is inappropriate for materials typically
9.2 Thetestmaterialshallbeatthesametemperatureasthat
producing death two or more days after administration of the
of the room in which the test is conducted at the time of
test compound unless the observation time between dosages is
administration to the animals.
increased. This test method can be successfully applied,
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
potential hazard to humans when pesticides, other chemicals, 10.2 Weight of each rat and calculate the dose according to
thisbodyweighttogivethespecifiedquantityoftestsubstance
or mixtures are ingested.
per unit of body weight.
5.4 This test method allows for observation of signs of
toxicityinadditiontomortality.Thisinformationcanbeuseful
10.3 Record all information necessary to document animal
in planning additional toxicity testing.
weights and volume of test substance administered to each
animal.
6. Apparatus
10.4 Dose four to six animals in the first stage each at a
6.1 Syringe, and an oral dosing needle or rubberized cath-
differentdosespanningtheestimateddoseresponsecurve(0%
eter to gavage the test compound is required.
lethality to 100 % lethality). Gavage animals using an oral
dosing needle or rubberized tubing. Observe each animal for a
7. Test Animals
minimum of 24 h. Use the methods of Feder et al (1992) to
7.1 Albino female rats weighing 190 to 300 g prefasted are
estimate the dose response curve and its parameters. Check to
used. A non in-bred rat such as the Sprague-Dawley strain is
see if the stopping rule (defined in 4.2) is met. If not, then
generally preferred. Female rats are preferred because histori-
proceed to the second stage of dosing. Return the animals to
cal data indicate that females in most instances have lower
either ad libitum or 2 to 3-h feeding immediately after dosing.
LD50 values than males.
Return the animal to either ad libitum or 2 to 3-h feeding
7.2 An additional test may be conducted with male rats, b
...