GB 15193.30-2025National food safety standard - Test of developmental neurotoxicity (English PDF)

Foreword

This document was issued on 2 September 2025 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 2 March 2026.

It is a GB standard without the /T suffix: compliance is mandatory in China.

1 Scope

GB 15193.30-2025 is the Chinese national food safety standard for the developmental neurotoxicity test. It is one of the most demanding studies in the toxicology battery: the substance is given to the dam through gestation and lactation, and the offspring are then followed through development with behavioural, sensory, motor and learning assessments before their brains are examined morphometrically. The reason for the effort is that the developing nervous system can be damaged at doses that produce no effect in an adult animal and no visible malformation, so nothing else in the battery would detect it. The standard sets the purpose and principle of the test, the instruments and reagents, the test methods, the observation indicators, the pathological examination, the processing of the data and the evaluation of the result, and the test report. It takes effect on 2 March 2026.

This Standard specifies the basic test methods and technical requirements for developmental

neurotoxicity.

This Standard is applicable to the evaluation of the developmental neurotoxicity of test

substances.

2 Terms and Definitions

2.1 Developmental neurotoxicity

Abnormal changes in the structure and function of the nervous system caused by exposure to

the test substance during development. These changes may occur at any stage of the life cycle.

2.2 Maternal Toxicity

Direct or indirect health damage caused by the test substance in pregnant female parent animals,

manifested as reduced weight gain, functional abnormalities, signs of poisoning, and even death,

etc.

2.3 No observed adverse effect level (NOAEL)

The maximum dose or concentration under specified test conditions, using existing techniques

or detection indicators, at which no adverse effects related to the exogenous chemical were

observed.

2.4 Lowest observed adverse effect level (LOAEL)

The lowest dose or concentration of an exogenous chemical that causes a certain effect in an

organism, compared to a suitable control, under specified test conditions.

3 Purpose and Principle of the Test

A test that exposes pregnant and lactating experimental animals to the test substance to evaluate

the neurotoxicity of their offspring. It typically observes changes in the structure and function

of the nervous system, as well as abnormalities in neurobehavior. This test can evaluate the

potential effects of the test substance on the functional and/or morphological development of

the nervous system in F1 generation animals; and determine whether the test substance has

developmental neurotoxicity and its NOAEL and/or LOAEL.

4 Instruments and Reagents

4.1 Instruments/apparatus

Rat jumping box, rat dark-light box, rat shuttle box, rat Morris water maze, tuning fork,

forelimb suspension device, hot plate, rat treadmill, righting reflex tester, commonly used

laboratory dissection instruments, electronic balance, biological microscope, ophthalmoscope,

biochemistry analyzer, blood analyzer, coagulation analyzer, centrifuge, pathological slicer, etc.

4.2 Reagents

Formaldehyde, xylene, ethanol, hematoxylin, eosin, paraffin, blood cell analyzer diluent,

biochemistry analysis reagents, coagulation analysis reagents, etc.

5 Test Methods

5.1 Test substance

The test substance shall be the original sample whenever possible. If the original sample cannot

be used, the test substance shall be appropriately processed according to GB 15193.21.

5.2 Laboratory animals

5.2.1 Selection of animals

The selection of laboratory animals shall comply with the relevant provisions of GB14922.

Animals with proven sensitivity to the test substance shall be selected. Generally, rodents,

particularly rats, are preferred. Avoid using strains with low reproductive rates or high rates of

developmental defects. The gestation and lactation days mentioned in this Standard refer to

commonly used rat strains. If other species are used, the corresponding number of days shall

be selected, and their rationale shall be explained based on relevant toxicological, toxicokinetic,

and/or other data. The species, strain, source, microbiological grade, sex, weight, and age of

the laboratory animals shall be clearly indicated.

5.2.2 Number and sex of animals

To obtain basic experimental data that meet statistical requirements and accurately evaluate the

toxic effects of the test substance on the neurodevelopmental process of animals, at least 20

litters of F1 generation animals shall be obtained for each dosage group and control group.

Within 4 days after birth (Postnatal Days, PND) (the birthday is PND 0), litter standardization

shall be performed; and excess F1 generation animals from each litter shall be randomly

removed to ensure that the number of F1 generation animals in each litter is as consistent as

possible and shall not exceed the average number of F1 generation animals per litter (8~12

animals) of the selected animal strain. The number of females and males in each litter of F1

generation shall be as consistent as possible. Selective removal of F1 generation animals is not

allowed, such as removing F1 generation animals based on body weight. Before the formal

experiment, it shall be determined which F1 generation animals shall be used for the pre-

weaning experiment and which for the post-weaning experiment.

5.2.3 Animal preparation

Use healthy, unused laboratory animals (except for consolidation experiments). Before the

experiment, animals shall undergo environmental acclimatization and quarantine observation

in the laboratory animal house for at least 3~5 days. At the start of the experiment, the difference

in animal weight shall not exceed +/-20% of the average weight; and the weight shall be within

the normal range for that animal strain. If pregnant animals are purchased, ensure they

acclimatize for 2~3 days. Pregnant rats mated with the same male shall be distributed as evenly

as possible among the groups, and each animal shall be uniquely identified.

5.2.4 Animal housing environment

The housing conditions, drinking water, and feed for laboratory animals shall comply with the

relevant provisions of GB 14925 and GB 14924.Laboratory animals shall have free access to

food and water. Mating shall take place in suitable cages. After successful mating, animals shall

be transferred to a farrowing cage or a pregnant rodent cage for individual feeding within 15

days of gestation. Cages shall be strategically placed to minimize locational impact. Provide

suitable nesting material for pregnant animals nearing delivery. Animals shall be handled with

care during pregnancy to prevent abortion and minimize external stimuli such as noise.

5.3 Dosage and grouping

The experiment shall include at least three dose groups and one control group. Under conditions

permissible by the physicochemical and biological properties of the test substance, the highest

dose shall induce some maternal toxicity, such as clinical signs, decreased weight gain (not

exceeding 10%), and/or dose-response relationships in target organ toxicity, but without

causing death. Intermediate doses shall cause mild toxic reactions. And low doses shall not

cause any toxic reactions. The interval between dose groups should ideally be 2 ~ 4 times. If a

fourth dose group is required, a larger interval (e.g., >10 times) can be chosen.

5.4 Administration of the test substance

5.4.1 Mixing with feed or water

When administering the test substance by mixing it with feed or drinking water, the test

6 Observation Indicators

6.1 General toxicity test

Hematological and blood biochemical indicators will be measured at the end of the experiment.

Recommended hematological indicators include white blood cell count and differential (at least

three categories), red blood cell count, hemoglobin concentration, hematocrit, platelet count,

prothrombin time (PT), and activated partial thromboplastin time (APTT), etc. If there is an

impact on the blood system, reticulocyte count and bone marrow smear cytology shall also be

performed additionally. Blood biochemical indicators shall be measured through a blood test

on an empty stomach. The measured indicators shall include electrolyte balance; glucose, lipid,

and protein metabolism; liver (cells, bile ducts), and kidney function, etc. The test shall include

at least the following indicators. alanine aminotransferase (ALT), aspartate aminotransferase

(AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea, creatinine

(Cr), blood glucose (Glu), total protein (TP), albumin (Alb), total cholesterol (TC), triglycerides

(TG), chloride, potassium, and sodium. If necessary, the following indicators may be tested,

such as calcium, phosphorus, uric acid (UA), total bile acids (TBA), cholinesterase, sorbitol

dehydrogenase, methemoglobin, and hormones, etc. Additional tests shall be conducted based

on the toxicological characteristics or structure-activity relationship of the test substance.

6.2 Clinical observation on F0 generation animals

The health status of all F0 generation animals shall be observed at least once daily, including

morbidity and mortality. In addition, detailed clinical observations shall be conducted regularly

during the administration and observation periods of the test substance (at least two

observations each during the administration periods of the test substance in pregnant and

lactating animals), with at least 10 pregnant and lactating animals observed in each dose group.

Blinding shall be used to observe the animals to reduce animal stress and observational bias;

and each study shall be conducted by a dedicated specialist. Physical signs of poisoning shall

be observed and recorded, including. coat, skin, eyes, mucous membranes, secretions, and

changes in autonomic nervous system activity (such as tearing, erect hair, abnormal breathing

such as open-mouth breathing, abnormal pupil size, and abnormal defecation and urination).

Other abnormal reactions such as body position, activity level (such as changes in exploratory

activities), and motor coordination shall also be observed and recorded. Record abnormal gait

(e.g., staggering, ataxia), posture (e.g., arched back), response to touch or environmental

changes, spasms or tonic movements, convulsions, tremors, lethargy, bizarre behaviors (e.g.,

biting, excessive grooming, abnormal head movements, repetitive circling, self-mutilation,

retreating, and vocalizations), or aggressive behaviors, etc. Record the onset date, time, severity,

and duration of physical signs of poisoning.

The weight of F0 generation animals shall be recorded near parturition or on the day of

parturition, and at weaning (PND 21). During gestation and lactation, record weekly feed

consumption. For F0 generation animals administered the test substance via drinking water,

record weekly water intake. Record changes in the weight of F0 generation animals; record

weight at least once a week during exposure, and at least twice a week for animals administered

via gavage.

6.3 Clinical observation on F1 generation animals

6.3.1 Requirements for clinical observation on F1 generation animals

The health status of all F1 generation animals shall be observed at least once daily, including

morbidity and mortality. Detailed clinical observation shall be conducted on F1 generation

animals during the administration and observation periods of the test substance, with the same

observation and recording requirements as in 6.2.

6.3.2 Physical development indicators on F1 generation animals

Record body weight according to Table 4 and observe changes in developmental indicators.

Based on the expected results and previous measurements, physical development indicators of

F1 generation animals can be measured multiple times during other developmental stages. No

test shall be performed within 2 days after weaning; relevant indicators can be tested earlier.

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