GB/T 35515-2017 English PDF

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GB/T 35515-2017: Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
Status: Valid

Standard ID	USD	BUY PDF	Lead-Days	Standard Title (Description)	Status
GB/T 35515-2017	759	Add to Cart	6 days	Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition	Valid

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Basic data

Standard ID: GB/T 35515-2017 (GB/T35515-2017)
Description (Translated English): Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition
Sector / Industry: National Standard (Recommended)
Classification of Chinese Standard: A80
Classification of International Standard: 13.300; 13.020.40
Word Count Estimation: 38,321
Date of Issue: 2017-12-29
Date of Implementation: 2018-07-01
Issuing agency(ies): General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of the People's Republic of China

GB/T 35515-2017: Chemicals -- Fish assay for oestrogenic and androgenic activity, and aromatase inhibition

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Chemicals-Fish assay for oestrogenic and androgenic activity, and aromatase inhibition ICS 13.300; 13.020.40 A80 National Standards of People's Republic of China Chemical fish estrogens, androgens and aromatase Inhibitory activity test method Published on.2017-12-29 2018-07-01 Implementation General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China China National Standardization Administration released Directory Preface III Introduction IV 1 Scope 1 2 Terms, definitions and abbreviations 1 3 Method Overview 1 4 Quality Assurance and Quality Control 2 5 Equipment 2 6 Test preparation 2 6.1 Test Water 2 6.2 Domestication of fish 2 6.3 Pre-exposure and fish selection 3 7 Test procedure 3 7.1 General instructions 3 7.2 Selection of test concentration 3 8 Test procedure 3 8.1 Selection and weighing of test fish 3 8.2 Exposure conditions 3 8.2.1 Time 3 8.2.2 Domestication 3 8.2.3 Lights and Temperature 4 8.3 Test frequency 4 8.4 Observation 4 8.4.1 Mortality 4 8.4.2 Behavior and appearance 4 8.4.3 Fish euthanasia 4 8.4.4 Observing Secondary Characteristics 4 8.4.5 Determination of vitellogenin 5 9 Data and Reports 5 9.1 Evaluation of Biomarker Response Using ANOVA 9.2 Test Result Report 5 Appendix A (informative) Sample collection procedure for vitellogenin analysis 7 Appendix B (informative annex) Assessment of the detection of secondary sexual characteristics of blackheadhead and barley with specific endocrine-active substances16 Appendix C (Informative Appendix) Experimental Conditions for Fish Endocrine Screening Test 21 Appendix D (informative) Spawning substrates for zebrafish and blackheaded fish23 Appendix E (Informative) Some Chemical Characteristics of Acceptable Dilution Water 25 Appendix F (Informative Appendix) Analysis of Yolk Protein Addition and Inter-Platform Standards 26 Appendix G (Informative) Statistical Analysis Process 27 Appendix H (Normative Appendix) Standards for Interpretation and Acceptability of Test Results 28 References 29 Figure A.1 Cut along the pectoral fins with scissors 8 Figure A.2 Using scissors along the abdomen line into the abdomen about 2mm from the head of the anus Figure A.3 Using surgical forceps to open the abdominal wall, exposing the liver and other internal organs (or securing the abdominal wall laterally) 9 Figure A.4 Slowly separating and resecting the liver with forceps 10 Figure A.5 Slowly removing the intestine with surgical forceps 10 Figure A.6 End of the bowel and any mesentery appendages cut with scissors 11 Figure A.7 (female) Same for the female fish process 11 Figure A.8 Complete the entire process 12 Figure A.9 Segmentation of zebrafish fish head 15 Figure B.1 Number and size of black-headed fish beads 17 Figure B.2 Gender Differences in Anal Fin Shape and Size 19 Figure B.3 Protuberance process on the joint plate of the hip fin line 20 Figure B.4 Fish with cutting points Photo 20 Figure D.1 Zebrafish Spawning Substrates 23 Figure D.2 Blackhead spawning substrate 24 Figure G.1 Statistical analysis flow chart 27 Table B.1 Blackhead Fish Star Template 18 Table C.1 Experimental conditions for the fish endocrine screening test 21 Table E.1 Some Chemical Characteristics of Acceptable Dilution Water 25

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009. The technical content of this standard and the Organization for Economic Cooperation and Development (OrganizationforEconomicCooperationandDevelopment, OECD) Test Guideline 230 (2009) "21d Fish Test. Short-Term Screening Test for Estrogen, Androgen and Aromatase Inhibition Activity" (English) Consistent. This standard is proposed and managed by the National Standardization Technical Committee for Hazardous Chemicals Management (SAC/TC251). This standard was drafted by. China Academy of Inspection and Quarantine, Guangdong Province Microbial Analysis and Detection Center, Shanghai Testing Center, Ningbo Entry Inspection and Quarantine Bureau. The main drafters of this standard are. Cui Yuan, Chen Huiming, Zeng Guofu, Li Haishan, Xie Wenping, Mei Chengfang, Yin Haowen, Liang Yihuai, and Chen Xiaoqing.

Introduction

This test method describes an in vivo screening test that exposes sexually mature male fish to females in the oviposition phase to chemistry 21d in the product. At the end of the 21d exposure period, the end points of the biomarkers for male and female fish were determined and used as test chemicals for estrogen stimulation. Prime, aromatase inhibition or androgenic activity indicators. These test endpoints include vitellogenin and secondary sexual characteristics. For black-headed octopus, Japanese green pheasant And zebrafish can be assayed for vitellogenin, while secondary sexuality can only be determined on blackheaded and Japanese barley. Vitellogenin is usually stimulated by circulating endogenous estrogens and is produced by the liver of female oviparous vertebrates. Vitellogenin is The precursor of vitellin, once produced in the liver, enters the ovary from the blood stream and is absorbed and modified by the developing egg. The vitellogenin is incomplete The plasma of the cooked females and males was barely detectable because they lacked enough circulating estrogen but were stung by exogenous estrogens. When stimulated, the liver can synthesize and secrete vitellogenin. The determination of vitellogenin can detect different estrogenic modes of action for chemicals, and the detection of estrogens may pass The production of vitellogenin in males was measured. Reduction of estrogen circulating levels in females, eg by inhibiting the conversion of endogenous males An aromatase that is a natural estrogen 17β-estradiol causes a decrease in vitellogenin levels, which is used to detect aromatase Inhibiting properties of chemicals. Use standardized tests that have been standardized. Use of immunochemical, species-specific enzyme-linked immunosorbent assay (ELISA) assays In the method, the blood of blackheaded fish, the homogenate of the blood or head/tail of the zebrafish, and the liver of the barley were collected as a sample for the measurement of VTG. for Barley, VTG measured in the blood has a good correlation with the liver. Appendix A provides analysis of vitellogenin Sample collection recommended procedure. The vitellogenin can be tested by different methods, but all are based on validated species-specific ELISA. method. The secondary sexual characteristics of males of specific species are observable and quantitatively responsive to endogenous androgen circulating levels. These characteristics are in blackheads. Fish and barley can be reflected, but the zebrafish is not reflected because the zebrafish does not have a quantifiable secondary sexuality. For blackheaded fish, the main indicator of exposure to exogenous substances is the number of bead stars located on the female's lips. For barley, the number of mastoids The major markers of exogenous exposure to androgens in females were composed. Appendix B illustrates the use of blackheaded fish and barley The process of evaluating sexual characteristics. Chemical fish estrogens, androgens and aromatase Inhibitory activity test method

1 Scope

This standard specifies the methodological overview, quality assurance, and quality of test methods for the inhibition of estrogenic, androgen and aromatase activities of chemicals in fish Controls, equipment, test preparation, test procedures, test procedures, data, and reports. This standard applies to the test of inhibitory activity of estrogens, androgens and aromatase enzymes in fish. 2 Terms, definitions and abbreviations 2.1 Terms and Definitions The following terms and definitions apply to this document. 2.1.1 Loading rate loadingrate The wet weight of a fish in a unit volume of water. 2.1.2 Carrying density stockingdensity The number of fish per unit volume of water. 2.1.3 Vitellogenin vitelogenin;VTG A phospholipid glycoprotein, a precursor of yolk protein, which is commonly found in sexually mature female individuals of all oviparous animals. 2.1.4 Maximum tolerated concentration maximumtoleratedconcentration;MTC The test article causes the highest test concentration of less than 10% mortality. 2.2 Abbreviations The following abbreviations apply to this document. ELISA. enzyme-linked immunosorbent assay

3 Method Overview

Females and males in the breeding period were co-exposed to the test substance. According to the selected test fish species its relevant biomarker endpoint Indicators are measured. Among them, sexual characteristics can be visually confirmed after dissection. For other biological test indicators, refer to Appendix C, Table C.1. Set 3 Concentration of test substance and a blank control, if necessary, solvent control. For barley and zebrafish, at least 2 per test concentration Test containers (5 male fish and 5 female fish per test container); for blackheads, at least 4 test concentrations per test concentration (2 male fish and 4 female fish are included in each test vessel). Exposure was conducted for 21 days and fish were sampled on Day 21 exposure. On the 21st day, all animals were euthanized. For the detection of secondary sexual characteristics of black-headed finfish and barley, refer to Appendix B; Blood samples of zebrafish and blackheaded fish, or zebrafish head/tail tissue homogenate samples for detection of vitellogenin, see Appendix A; for blue ......

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