GB/T 15670.15-2017Toxicological test methods for pesticides registration - Part 15: In vivo mammalian bone marrow polychromatic erythrocyte micronucleus test (English PDF)

Foreword

GB/T 15670 Toxicological Test Methods for Pesticides Registration may be divided into the following parts:

--- Part 1: General Principles;

--- Part 2: Acute Oral Toxicity Test - Horn's Method;

--- Part 3: Acute Oral Toxicity Test - up-and-down-Procedure;

--- Part 4: Acute Oral Toxicity Test - Miller and Taninter's Method;

--- Part 5: Acute Dermal Toxicity Test;

--- Part 6: Acute Inhalation Toxicity Test;

--- Part 7: Dermal Irritation/Corrosion Test;

--- Part 8: Acute Eye Irritation/Corrosion Test;

--- Part 9: Skin Sensitisation Test;

--- Part 10: Short-Term Repeated Dose 28-day Oral Toxicity Study;

--- Part 11: Short-Term Repeated Dose 28-day Dermal Toxicity Study;

--- Part 12: Short-Term Repeated Dose 28-day Inhalation Toxicity Study;

--- Part 13: Sub-chronic Toxicity Study;

1 Scope

GB/T 15670.15-2017 is the Chinese national standard on toxicological test methods for pesticides registration - part 15: in vivo mammalian bone marrow polychromatic erythrocyte micronucleus test, in the field of agriculture. The /T suffix marks it as a recommended standard: it is not compulsory by itself, but it becomes binding as soon as a contract, a tender or a customer specification calls it up - which in practice is how most foreign buyers meet it. It was issued on 12 July 2017 by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China; Standardization Administration of China, and has been in force since 1 February 2018. Classification: ICS 65.100, CCS B17. This page is published from the official record of the standard held by the Chinese standards administration: the identification, the dates, the classification and the issuing body are taken from there. The clause text, the tables and the numeric limits are in the document itself, which is delivered complete in English translation.

This Part of GB/T 15670 specifies the basic principles, methods and requirements of the micronucleus test of mammalian bone marrow polychromatic erythrocyte in vivo. This Part is applicable to the micronucleus test of mammalian bone marrow polychromatic erythrocyte in vivo for pesticides registration.

2 Normative References

The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) is applicable to this document.

GB 14925 Laboratory Animal - Requirements of Environment and Housing Facilities

3 Terms and Definitions

For the purpose of this document, the following terms and definitions apply.

3.1 Micronucleus In the late stage of cell mitosis, fragments of chromosomes or delayed chromosomes that cannot enter the nucleus of the daughter cells, one or several secondary nuclei formed in the cytoplasm of the daughter cells, which are consistent with the coloration of the main nucleus, and are round or oval.

3.2 Polychromatic erythrocytes; PCE Immature erythrocytes are an intermediate stage in the process of erythrocyte maturation. At this time, because the cytoplasm still contains ribosomes, it remains basophilic for about 24h, which can be distinguished from mature erythrocytes by selective staining.

6.1.2.1 Instruments: biological microscope, constant temperature water bath, etc.

6.1.2.2 Calf serum (inactivated): After filtering the bacteria, calf serum is placed in a constant temperature water bath at 56°C for 30min for inactivation. Inactivated calf serum is usually stored in a refrigerator at 4°C. Rat and mouse serum can also be used instead.

6.1.2.3 Giemsa dye solution: Giemsa dye: 3.8g Methanol: 375mL Glycerol: 125mL Preparation: place Giemsa dye and a small amount of methanol in a mortar and grind carefully; then add methanol to 375mL; after it is completely dissolved, add 125mL of glycerol and mix well. Put it in a 37°C thermostat for 48h. Shake several times during the heat preservation period to promote the full dissolution of the dye. Take out and filter; and use it after two weeks.

6.1.2.4 Phosphate buffer solution (pH 6.8): 1/15mol/L disodium hydrogen phosphate solution: take 9.47g of Na2HPO4 and dissolve it in 1000mL of distilled water. 1/15mol/L potassium dihydrogen phosphate solution: take 9.07g of KH2PO4 and dissolve it in 1000mL of distilled water. Mix 50 mL of disodium hydrogen phosphate solution with 50 mL of potassium dihydrogen phosphate solution; measure and adjust to pH

6.8 with a pH meter.

6.1.2.5 Giemsa application solution: take 1 portion of Giemsa dye solution and 6 portions of phosphate buffer solution (pH 6.8); mixed and prepared for current use.

6.1.2.6 Methanol (analytically pure).

6 Test Methods...

8 Test Report

The test report shall include at least the following:

a) Test name, test organization name and contact information, report number;

b) The name and contact information of the test entrusting organization, the date of sample acceptance and the sample sealing situation;

c) The start and end date of the test, the person in charge of the test project, the technical person in charge of the test organization, and the date of issuance;

d) Test summary;

e) Name of test substance, active ingredient American Chemical Abstracts registration number (CAS number) (if known), code (if any), purity (or content), dosage form, production date (batch number), physical and chemical properties, and solvent used for preparation and preparation method;

f) The species, strain, grade, quantity, weight, gender, source (supplier name, laboratory animal quality certificate number, laboratory animal production license number), quarantine, adaptation of the laboratory animal. Breeding environment of the laboratory animal, including temperature, relative humidity, feed, single cage breeding or group breeding, laboratory animal facility use permit number;

g) Dose and group, including the principle or basis for selecting the dose, dose and group, animal grouping method and the number of animals of each gender in each group;

h) Test conditions and methods, including main instruments and equipment, route of exposure, exposure plan, operation steps, name of positive control substance, etc.;

i) Test results: summarize item by item with text description and table, poisoning performance, mean and standard deviation of polychromatic erythrocytes with micronuclei in each group, proportion of polychromatic erythrocytes in total erythrocytes, dose-response relationship, statistical analysis;

j) Test conclusion: give a conclusion on whether the test substance has mutagenic effects under the test conditions, and discuss relevant issues when necessary;