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GB 15193.3-2014: National Food Safety Standard -- Acute toxicity test
GB/T 15193.3-2014
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Acute toxicity test
ISSUED ON: DECEMBER 24, 2014
IMPLEMENTED ON: MAY 01, 2015
Issued by: Health and Family Planning Commission of PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions... 4 
3 Test purpose and principle ... 4 
4 Test methods ... 5 
5 Data processing and result evaluation ... 14 
6 Report ... 14 
7 Interpretation of the test ... 15 
Appendix A Calculation of LD50 value in Horn's method (calculation table for the
determination of LD50 by dose escalation method) ... 16 
Appendix B Dose gradient table for up-down procedure (UDP) with different slopes
... 21 
Appendix C Calculation of LD50 point estimates and confidence limits for formal test
... 23 
Appendix D Response rate-probability unit table ... 25 
Appendix E Probability units equivalent to 0% and 100% response rates ... 26 
Appendix F Observation items in experimental animal poisoning performance ... 27 
Appendix G Dose grading of acute toxicity (LD50) ... 28 
National food safety standard - Acute toxicity test
1 Scope
This standard specifies the basic test methods and technical requirements, for acute oral
toxicity test.
This standard applies to the evaluation of the acute oral toxicity of the test substance.
2 Terms and definitions
2.1 Acute oral toxicity
The toxic effects on animals, in a short period of time, after one or more oral
administration of the test substance to experimental animals within 24 h.
2.2 The half lethal dose (LD50)
The dose of the test substance, that can cause animal mortality to be 50%, after oral
administration of the test substance once or multiple times within 24 h. It is the
calculated value, which is obtained through statistics. Its unit is the number of
milligrams or grams of the test substance, which is ingested per kilogram of body
weight, that is, mg/kg body weight or g/kg body weight.
3 Test purpose and principle
The acute oral toxicity test is the most basic test, for detecting and evaluating the toxic
effects of the test substance, that is, after the test substance is administered orally once
or multiple times within 24 hours, the toxic reactions produced by animals, including
poisoning sign and death, are observed in a short period of time. It is usually expressed
as LD50.
This test can provide information on health hazards, which are caused by oral exposure
to the test substance in the short term. It serves as the basis for acute toxicity grading.
It provides the basis for dose selection and observation indicators for further toxicity
tests. It preliminarily estimates the target organs of toxic effects AND the possible toxic
action mechanism.
4 Test methods
4.1 Test substance
4.1.1 Preparation of test substances
It shall dissolve or suspend the test substance in a suitable solvent. The preferred solvent
is water. The test substance, which is insoluble in water, can use vegetable oil (such as
olive oil, corn oil, etc.). The test substance, which is insoluble in water or oil, can also
use carboxymethyl cellulose, starch, etc., to prepare a suspension or paste. The test
substance shall be freshly prepared, unless there is data showing that its solution or
suspension is stable during storage.
4.1.2 Administration of test substance
4.1.2.1 Approach
Oral gavage.
4.1.2.2 Fasting before the test
Animals need to fast before the test. Generally, rats need to fast overnight (generally
about 16 h); mice need to fast for 4 h ~ 6 h; BUT they can drink water freely. After
administration of the test substance, rats need to continue fasting for 3 h ~ 4 h; mice
need to continue fasting for 1 h ~ 2 h. If the test substance is administered in batches
multiple times, the animals can be given a certain amount of feed, according to the
length of the exposure interval.
4.1.2.3 Gavage volume
The gavage volume of each test substance group shall be the same, which is 10 mL/kg
body weight for rats AND 20 mL/kg body weight for mice. If the solvent is water, the
maximum gavage volume of rats can reach 20 mL/kg body weight and that of mice can
reach 40 mL/kg body weight.
4.1.2.4 Method
The test substance is generally administered at one time. It can also be administered
multiple times in one day (each interval is 4 h ~ 6 h, no more than 3 times within 24 h,
the maximum dose is reached as far as possible, which is calculated as a single dose).
4.1.2.5 Observation period
General observation period is 14 d, which may be extended to 28 d if necessary. It shall
be observed for at least 7 d, in special emergency situations.
4.2 Experimental animals
4.2.1 Animal selection
The selection of experimental animals shall comply with national standards and
relevant regulations (GB 14923, GB 14922.1, GB 14922.2). Choose healthy adult rats
(180 g ~ 220 g) and/or mice (18 g ~ 22 g) of two genders, OR choose other experimental
animals. Females shall be unmated and unpregnant. The difference in body weight,
between animals of the same gender, shall not exceed ±20% of the average body weight.
4.2.2 Animal preparation
Before the test, the experimental animals shall undergo at least 3 d ~ 5 d environmental
adaptation and quarantine observation, in the test environment.
4.2.3 Animal feeding
The feeding conditions, drinking water, feed of experimental animals shall comply with
national standards and relevant regulations (GB 14925, GB 5749, GB 14924.1, GB
14924.2, GB 14924.3). Animals in each test group are raised in separate cages by gender.
The number of animals per cage shall not affect the free movement of animals AND it
can observe the physical signs of animals. For the special biological characteristics and
toxic reactions (such as irritability, cannibalism, etc.), which are often caused by some
test substances, it can be raised in a single cage. During the test, the experimental
animals are fed with basal feed AND allowed for free access to water.
4.3 Design methods for several commonly used acute toxicity tests
4.3.1 Horn's method
4.3.1.1 Pre-test
According to the nature of the test substance and the known data, the following methods
are used: Generally, it uses the dose of 100 mg/kg body weight, 1000 mg/kg body
weight, 10000 mg/kg body weight, each of which uses 2 ~ 3 animals for pretest.
According to the death situation within 24 h, estimate the possible range of LD50.
Determine the dose group of the formal test. A single dose, such as 215 mg/kg body
weight, can also be simply used directly in a pretest, with 5 animals. Observe the
poisoning performance of animals, within 2 hours. If the signs of poisoning are severe,
it is estimated that most animals may die, THEN the dose series below 215 mg/kg body
weight can be used to perform the formal test. Otherwise, the dose series above this
dose can be used if the signs of poisoning are mild. Pretests are not required, if there
are relevant documents.
4.3.1.2 Formal test
4.3.1.2.1 Number of animals
Generally, there are 10 animals in each group, half male and half female.
4.3.3.1 Scope of application
This method is mainly applicable to the test substance, which has high purity, high
toxicity, low intake, AND the animal will die, within 1 d ~ 2 d after administration of
the test substance. For the test substance, which causes the animal to die at 5 d and
above, after administration, it is not applicable. The test can...