GB 15193.3-2014National food safety standard - Acute toxicity test (English PDF)

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.

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.

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.

4.1.2.5 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.

4.3 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.

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.

4.3.2 Limit test

4.3.2.1 Scope of application

This method is applicable to the test substance, whose data shows little or no toxicity.

When the animal is administered with a certain dose of the test substance, there is still

no death.

4.3.2.2 Number of animals

It generally selects 20 animals, half male and half male.

4.3.3 Up-down procedure (UDP)

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 be carried out, according

to the dose sequence selected by the tester OR under the guidance of a special software

package; the test results can be counted.

4.3.4 Korbor method

4.3.4.1 Pre-test

Unless otherwise required, the dose, at which all animals die or more than 90% of the

animals die, AND the dose, at which no animals die or less than 10% of the animals die,

shall be obtained in the pre-test, as the highest dose and the lowest dose in the formal

test, respectively.

4.3.6 Acute combined toxicity test

4.3.6.1 Principle

When two or more test substances exist at the same time, three different combined

action modes of antagonism, addition or synergy may occur. These three different

actions can be determined, according to certain formulas and judgement criteria.

4.3.6.2 Procedure

4.3.6.3 Calculation

4.3.6.3.1 Each test substance in the mixture is mixed in an equal toxic ratio, so the

calculated LD50 is multiplied by the ratio of each test substance, to obtain the dose of

each test substance.

4.4 Observation indicators

4.4.1 Clinical observation

Observations include skin, hair, eyes, mucous membranes, respiratory system,

genitourinary system, digestive system, nervous system, etc. Special attention shall be

paid to the presence or absence of tremor, convulsions, salivation, diarrhea,

sluggishness, lethargy, coma (see Appendix F).

4.4.2 Pathological examination

All animals, including those died during the test, humanely sacrificed, sacrificed at the

end of the test, shall undergo gross anatomical examination. Record the gross

pathological changes of each animal. It shall perform histopathological observation,

where there are gross anatomical pathological changes.

6.1 Test name, test organization name, contact information, report number.

6.2 The name and contact information of the test entrusting organization, as well as the

date of sample acceptance.

6.3 The start and end date of the test, the person in charge of the test item, the technical

person in charge of the test organization, the date of issue.

6.4 Test summary.

6.5 Test substance. Name, batch number, dosage form, state (including sensory,

properties, packaging integrity, identification), quantity, pre-treatment method, solvent.

6.8 Test results. Animal growth and activity, body weight, body weight gain, response

of each animal (including signs of toxicity, severity, duration, reversibility, death rate

list, gross anatomy of each animal, histopathological findings, LD50 and 95%

confidence limits, statistical treatment method of the results.