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GB/T 21773-2025: Chemicals - Test method of in vivo mammalian erythrocyte micronucleus
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GB/T 21773-2025: Chemicals - Test method of in vivo mammalian erythrocyte micronucleus

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ICS 13.300 CCSA80 National Standards of the People's Republic of China Replaces GB/T 21773-2008 Chemicals in mammalian red blood cells Micronucleus test method Published on 2025-08-29 Implemented on December 1, 2025 State Administration for Market Regulation The State Administration for Standardization issued a statement.

Foreword

This document is in accordance with GB/T 1.1-2020 "Standardization Work Guidelines Part 1.Structure and Drafting Rules of Standardization Documents". Drafting is scheduled. This document supersedes GB/T 21773-2008 "Test Method for Micronuclei in Mammalian Red Blood Cells of Chemicals", and is consistent with GB/T 21773- Compared to.2008, aside from structural adjustments and editorial changes, the main technical changes are as follows. a) The content of the "Basic Principles of Experimentation" has been revised (see Chapter 4, Chapter 3 of the.2008 edition); b) A new chapter on "Laboratory Proficiency Testing" has been added (see Chapter 5); c) The sections on “Animal Selection,” “Housing Conditions,” “Animal Preparation,” and “Test Substance Preparation” have been revised (see 6.1, 4.1 of the.2008 edition); d) The contents of "Solvent/Excipients" and "Control" have been changed (see 6.2, 4.2 of the.2008 version); e) The content regarding "Animal Number and Sex" (see 6.3.1, 4.3.1 in the.2008 version) and the content regarding "Dose Level" (see...) have been modified. 6.3.2 (4.3.3 in the.2008 edition), the content of "Limited Quantity Testing" (see 6.3.3, 4.3.4 in the.2008 edition), and the content of "Route of Exposure" The contents of the “Discharge Procedure” (see 6.3.4, 4.3.5 of the.2008 edition) and the “Discharge Procedure” (see 6.3.5, 4.3.2 of the.2008 edition); f) Added sections on "Observation" (see 6.3.6) and "Target Tissue Exposure" (see 6.3.7); g) The content of "Bone Marrow/Blood Sample Preparation" (see 6.3.8, 4.3.6 in the.2008 version) and "Analysis (Manual and Automated)" have been changed. The content of "Analysis" (see 6.3.9, 4.3.7 of the.2008 edition); h) The content of "Data Processing" has been changed (see 7.1, 5.1 in the.2008 version); i) Added content on "Quality Control" (see 7.2); j) The content of "Results Evaluation and Interpretation" (see 7.3, 5.2 of the.2008 version) and the content of "Trial Report" (see 7.4,.2008 version) have been revised. (5.3 of the.2018 edition). Please note that some content in this document may involve patents. The issuing organization of this document assumes no responsibility for identifying patents. This document was proposed and is under the jurisdiction of the National Technical Committee on Standardization of Hazardous Chemicals Management (SAC/TC251). This document was drafted by. the National Institute for Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention; and the National Institute for Occupational Health and Related Product Safety, Chinese Center for Disease Control and Prevention. Institute of Health and Poison Control, Hunan Provincial Institute of Occupational Disease Prevention and Treatment. The main drafters of this document are. Dai Yufei, Shen Meili, Nie Yunfeng, Chen Yuanyuan, Li Li, Deng Fuchang, Gu Wen, and Liu Shuai. The release history of this document and the document it replaces is as follows. ---First published in.2008 as GB/T 21773-2008; ---This is the first revision.

introduction

The in vivo mammalian erythrocyte micronucleus assay is an in vivo genotoxicity test that detects erythrocyte micronuclei in the bone marrow of animals (usually rodents). Or assess the formation of micronuclei in peripheral blood erythrocytes to determine if chemicals have caused damage to erythroblast chromosomes or mitotic apparatus. Micronuclei The purpose of the experiment is to identify chemicals that can cause cellular genetic damage, leading to the formation of micronuclei, which may contain delayed chromosomes. Chromosome fragments or entire chromosomes. Micronucleus assays in mammals are of particular significance in assessing the genotoxicity of chemicals, although there may be [missing information]. While species differences exist, this experiment can reflect the impact of processes such as metabolism, toxicokinetics, and the body's DNA repair capacity on genetic damage. The in vivo micronucleus assay can also be used to conduct further research on genotoxic substances detected in in vitro assays. During the development of erythroblasts in the bone marrow into immature erythroblasts at a specific stage, their nuclei are expelled, and the resulting micronuclei... They may remain in the cytoplasm. Because these cells lack a nucleus, micronuclei are easier to observe or detect. In animals exposed to chemicals, if... An increased micronucleated cell rate in immature red blood cells indicates that chemicals induced chromosomal structural or numerical aberrations. Newly formed micronucleated cells... After staining, red blood cells can be observed and counted manually using a microscope, or automated analysis methods can be used. Automated analysis systems can be used... The system can significantly improve counting efficiency and can replace manual counting. With proper calibration, the automated analysis method can be used both in-laboratory and inter-laboratory. The reproducibility and sensitivity are superior to manual counting methods. Automated analytical systems suitable for erythrocyte micronucleus assays include, but are not limited to. Flow cytometer, image analysis platform, and laser scanning cytometer. In this experiment, chromosome fragments can be distinguished from whole chromosomes using various criteria, such as identifying the presence of kinetochore or centromere DNA. Whether or not, both are characteristics of a complete chromosome. If no kinetochore or centromere DNA is detected in the micronucleus, it indicates that it contains only chromosomes. Fragments; if kinetochore or centromere DNA is detected in the micronucleus, it indicates that an entire chromosome has been lost. It should be noted that the above distinction between chromosome fragments... Methods involving chromosome fragments and whole chromosomes are typically not included as part of the test. The target tissue for this experiment is juvenile rodents. The bone marrow is the primary site of red blood cell production. If there is evidence that immature red blood cells in the peripheral blood of certain mammalian species are... The detection of chemically induced chromosomal structural or numerical aberrations is sensitive enough, and immature red blood cells in peripheral blood can also be used as the detection target. The micronucleated cell rate of immature erythrocytes in bone marrow or peripheral blood is the primary endpoint. If the spleen of a mammalian species contains micronucleated cells... If the clearance effect is weak, or if the experimental animals are continuously exposed to the virus for a period exceeding the lifespan of the species' erythrocytes (e.g., mice exposed to the virus for 4 weeks or more), then... In both cases, the micronucleated cell rate of mature erythrocytes in peripheral blood can be selected as the detection endpoint. Before obtaining toxicity assessment data for a mixture in accordance with this testing guideline and using it for intended regulatory purposes, it is necessary to assess whether it can provide [the necessary information]. This objective is to provide compliant results while analyzing the reasons that may affect the applicability of the results. If there are regulatory requirements for the testing of mixtures, then... The above assessment is unnecessary. Chemicals in mammalian red blood cells Micronucleus test method 1.Scope This document establishes the basic experimental principles for the in vivo mammalian erythrocyte micronucleus assay, and specifies the laboratory proficiency testing, experimental parameters, and procedures. The test methods were described in accordance with the requirements of the report. This document applies to the in vivo mammalian erythrocyte micronucleus assay for chemicals. This document does not apply to chemicals or their metabolites that have not reached the target tissue.

2 Normative references

The contents of the following documents, through normative references within the text, constitute essential provisions of this document. Dated citations are not included. For references to documents, only the version corresponding to that date applies to this document; for undated references, the latest version (including all amendments) applies. This document. GB 14925 Laboratory Animal Environment and Facilities 3.Terms and Definitions The following terms and definitions apply to this document. 3.1 centromere The region in a chromosome that binds two sister chromatids together. 3.2 Kinetochore A disc-shaped structure formed by various proteins at the centromere of chromosomes during cell division. 3.3 micronuclei During telophase of mitosis (or meiosis), chromosome fragments or entire chromosomes, formed by lagging chromosomes, separate from the main nucleus and exist independently. The small core. 3.4 mature erythrocyte Red blood cells that lose residual RNA and/or other short-term markers after enucleation. Note. It can be distinguished from immature red blood cells using selective ribosomal dyes. 3.5 Immature red blood cells in the process of development containing residual RNA. Note. It can be distinguished from mature red blood cells using selective ribosomal dyes. 3.6 reticulocyte An immature red blood cell that, after being stained with a live dye, exhibits residual RNA aggregation into a unique network structure. Note. Immature red blood cells, in a broad sense, include both nucleated and anucleated stages. The micronucleus assay primarily observes immature red blood cells without nuclei, i.e., polychromatic erythrocytes. Cells or reticulocytes. 4.Basic Principles of the Experiment Appropriate routes of exposure should be used to expose laboratory animals to the toxin. If bone marrow samples are used, humane treatment should be administered to the animals at an appropriate time after exposure. Euthanasia is performed, followed by bone marrow extraction, slide preparation, and staining. If peripheral blood samples are used, blood is collected at an appropriate time after exposure for slide preparation and... Staining. When performing acute drug exposure, the appropriate time for bone marrow or blood sample collection should be chosen to detect the presence of substances induced by drug exposure. Micronucleated immature red blood cells. Sufficient time should be allowed before collecting peripheral blood samples to ensure that micronuclei appear in the circulating blood. This can be detected by microscopy. Microscopic observation, image analysis, flow cytometry, or laser scanning cytometers are used to detect the presence of micronuclei in the slides. 5.Laboratory proficiency testing 5.1 Competency Survey Before using micronucleus detection methods for routine testing, laboratories should demonstrate the capability to reproduce the results in published data. Expected results include the use of at least two positive controls (including weak positive results from low-dose positive controls), such as the positive controls recommended in Table 1. The experiment should include a solvent/excipient control. The dosage used in the experiment should be repeatable and demonstrate a dose-response relationship. The detection method should be confirmed for its sensitivity and dynamic range in the target tissue (bone marrow or peripheral blood), and should be based on the evaluation criteria used by the laboratory. Analysis should be performed using separate methods. This requirement does not apply to laboratories with testing experience (i.e., laboratories with historical databases). 5.2 Historical Comparison Data 5.2.1 During the proficiency survey, the laboratory should establish the range and distribution of historical positive controls, as well as the range and distribution of historical negative controls. 5.2.2 When establishing a historical negative control distribution based on the initial data acquisition, if published control data exists, the parallel negative controls should be compared with the published data. The control data in the table should be consistent. If more experimental data are included in the historical control distribution, the parallel negative control data should be located within that distribution. Within 95% control limits. The laboratory's historical negative control database should be statistically robust to ensure that its negative control data can be evaluated. Cloth characteristics. The minimum requirement for establishing a historical control database in a laboratory is to conduct at least 10 experiments and accumulate corresponding data, but it is advisable to do so within comparable... At least 20 experiments should be conducted under the experimental conditions to further improve the statistical significance and reliability of the data. The laboratory should employ quality control methods. Methods, such as control charts, are used to identify data variability and demonstrate that the method is "controlled" in the laboratory. Historical data are established and used... For the requirements (such as inclusion and exclusion criteria for historical data, and acceptability criteria for specific trials), please refer to relevant literature. 5.2.3 During the proficiency survey, if the laboratory is unable to complete sufficient experiments to establish a statistically robust negative control distribution, it may do so in the first instance. The negative control results obtained during routine testing should be consistent with published negative control data. 5.2.4 When adjusting the experimental protocol, its impact on the experimental data should be assessed, and it should be ensured that it remains consistent with the laboratory's existing historical control database. Consistency. If experts confirm a significant difference between this database and previous data distributions, a new historical comparison database should be established. During the reconstruction process... In China, if a laboratory can demonstrate that its parallel negative control values are consistent with previous databases or corresponding published data, then it is not necessary to establish a complete [database/system]. A negative control database can be used to conduct actual tests. 5.2.5 Negative control group data should include the micronucleated cell rate of immature red blood cells for each experimental animal. Parallel negative control data should be located in... The data should be within the 95% control limit of the laboratory's historical negative control database distribution. If parallel negative control data exceed the above 95% control limit, but the data... Data that are not extreme outliers, whose detection methods can be verified to be "controlled," and which show no technical or human error, can be included in historical data. ...