Chemicals—Test method of in vitro skin corrosion—Human skin model test
Some standard content:
ICS 13. 300: 11. 100
National Standard of the People's Republic of China
GB/T27830--2011
Chemicals
In vitro skin corrosion
Test method of in vitro skin corrosion-Human skin model testIssued on 2011-12-30
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministration of Standardization of the People's Republic of China
Implementation on 2012-08-01
This standard was drafted in accordance with the rules given in GB/T1.1-2009GB/T27830-2011
This standard is consistent with the technical contents of the Organisation for Economic Co-operation and Development (OECD) Guide for Testing Chemicals No. 431 (2004) In vitro skin corrosion: In vivo skin model test\ (English version). This standard has been modified in the following aspects: a chapter on scope has been added; - the contents of "Introduction" and "Preliminary considerations" in the original text of OECD 431 are used as the introduction of this standard; - the "Definition" in the original appendix of OECD 431 is used as "2 Terms and Definitions" of this standard; - the Table 2 in the original text of OECD 431 is used as "Appendix A (Informative Appendix)" of this standard; - the measurement units are changed to the legal measurement units of my country. This standard was proposed by the National Technical Committee for Standardization of Hazardous Chemicals Management SAC/TC251 and drafted by the Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Guangdong Exit-Entry Inspection and Quarantine Bureau, China Institute of Inspection and Quarantine, and China Chemical Economic and Technological Development Center. The main drafters of this standard are Liu Qingjun, Wang Xiaobing, Ci Ke, Cheng Shujun, Yang Ting, Xu Chonghui, and Jiao Hong. TTTKANYKACA
GB/T 27830—2011
Skin corrosion refers to the irreversible tissue damage caused by skin contact with the test substance (according to the GHS definition [11, this standard provides a method for evaluating skin corrosion without the use of in vitro animals or animal tissues. Skin corrosion evaluation often requires the use of experimental animals [\1. Taking into account the pain and harm suffered by animals in experiments, in 2002, the revised version and annex of the ECD Test Guide & Acute Skin Irritation and Storage Corrosion of Chemicals\ (TG404) proposed to allow the use of in vitro alternative methods to test skin corrosion in order to avoid pain and harm to animals. my country GB/T21604200 8 This method is equivalent to the one adopted. The main difficulty in completely replacing the in vivo skin corrosion test of Guide 404 (or GB/T21601-2008) is the lack of formal, independent and validated in vitro test methods. For an alternative method to be used as a skin corrosion substitute test for regulatory purposes, the first step is to conduct a preliminary validation study, followed by a formal validation study to evaluate the in vitro skin corrosion test method [4-]. Based on the results of these studies and other published literature, the following tests are recommended for the evaluation of in vivo skin corrosion: human skin model test (see this standard) and transcutaneous resistance test (see TG430) 13). Validation studies have shown that the use of the human skin model test can well distinguish known skin corrosive substances from non-skin corrosive substances [3,. It can also provide information for distinguishing between severe and mild skin corrosion. The test method described in this standard can identify corrosive chemicals and mixtures. Combined with other existing information (such as pH, structure-activity relationship, human and/or animal data): 12.13147, through weight of evidence analysis, this standard can also further identify non-corrosive substances and mixtures. This standard generally cannot provide sufficient information on the skin irritation of the test substance, nor can it further classify corrosive substances according to the GHS system 12.1314].
In order to fully evaluate the local skin reactions after a single skin exposure, it is recommended to conduct experiments in accordance with the sequential test strategy provided by GB/T216042008° and GHS. The sequential test strategy includes in vitro skin corrosion (such as this standard) and skin irritation tests before considering live animal tests.
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1 Scope
In vitro skin corrosion of chemicals
Test methods for human skin model
GB/T 27830—2011
This standard specifies the terms and definitions, test principles, test methods and data and information for the test methods for in vitro skin corrosion of chemicals on human skin model:
This standard is applicable to the screening and detection of skin corrosion of chemicals, and can be used as one of the alternative methods for animal tests on skin corrosion of chemicals, and can also be used as a part of toxicity tests, combined with other tests for the overall evaluation of the toxicity of chemical substances. 2 Terms and definitions
The following terms and definitions apply to this document. 2.1
In vivo skin corrosion Irreversible damage caused by skin contact after 4 hours of testing, that is, visible lesions appear from the epidermis to the dermis. The characteristics of corrosive effects are: ulcers, bleeding, bloody crusts, and after 14 days, skin atrophy, local alopecia, and crusts. For coagulated skin lesions, histopathological examination should be performed.
Cell viability
An indicator for measuring the overall viability of a cell population [e.g., the ability of the cell mitochondrial dehydrogenase reduction dye thiazolyl blue (3-(4,5-dimethylthiazol-2-yl)-2,5-dithiazolidine salt, MTT) (A$N0.298-93-1): The value of the indicator depends on the test site and the protocol used, and is related to the total number of cells and/or the number of active cells. 3 Principle of the test
The test substance is applied topically to a three-dimensional human skin model, which is at least a reconstructed epidermis containing a functional stratum corneum. The corrosive substance is identified based on the fact that the test substance reduces the cell viability (e.g., measured by MTT reduction method) to a set value level within a specific exposure time. The principle of the human skin model test is based on the assumption that corrosive chemicals penetrate the stratum corneum through diffusion or erosion after exposure to the skin, and produce cytotoxic effects on the underlying cell layer. 4 Test method
4. 1 Human skin model
Human skin models can be constructed by oneself or obtained through commercial channels: 1a1 or developed and prepared by the laboratory [sD-21]. The use of human skin should meet national and international ethical requirements. Any new skin model should be validated (at least meet the requirements of 4.3). The human skin model used in this test should meet the requirements listed in 4.2 and 4,3. 4. 2 General conditions of skin model
4.2.1 Human keratinocytes should be used to construct the epithelium of the skin model, and the functional stratum corneum should contain multiple layers of functional epithelial cells1
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cells. The skin model can have a matrix layer. The stratum corneum should be a multi-layer structure containing essential lipid components to form a functional barrier to resist the rapid penetration of cytotoxic substances.
4.2.2 The closed properties of the model should be able to prevent substances retained in the stratum corneum from entering the active tissue. In an ideal skin model, there should be no channels for the test substance to enter the active tissue around the stratum corneum, otherwise it will not be able to simulate the actual situation of skin exposure. 4.2.3 The skin model should be protected from bacteria (including mycoplasma) and fungal contamination. 4.3 Functional conditions of the skin model
4.3.1 Cell activity range can be quantitatively determined using MTT or other metabolic conversion-related vital dyes. In the test, the optical density (D) value of the dye extracted or dissolved from the negative control skin tissue should be at least 20 times the D value extracted using solvent alone [21. The negative control skin tissue should be able to maintain stable culture during the test (tissue activity remains stable). 4.3.2 The stratum corneum should be able to fully prevent the rapid penetration of certain cytotoxic chemicals (such as 1% TritonX-100). This performance can be evaluated by the exposure time that reduces cell activity by 50% (ETs). 4.3.3 The skin elastic tissue is best to show good reproducibility between different laboratories and over a long period of time. Moreover, according to the selected test protocol, the skin tissue can predict the corrosion energy of the reference chemicals listed in Table A.1 of Appendix A. 4.4 Test substance treatment
4.4.1 Each treatment group (including the control group) uses two identical skin tissues. For liquid test substances, the amount of liquid used should be sufficient to cover the entire skin surface. The morning application is 25uF./cm. For solid test substances, the amount should be sufficient to evenly cover the skin surface. Deionized water or distilled water can be used to dissolve the substance to ensure good exposure to the skin. If conditions permit, solid test substances should be ground into powder before use. The treatment method should be suitable for the test substance. After treatment, use an appropriate buffer or 0.9% NaCl solution to carefully wash off the test substance on the skin surface.
4.4.2 Each test should have positive and negative control tests to ensure the normal operation of the test model. Glacial acetic acid or 8 mol/L potassium hydroxide (KOH) is recommended as a positive control. The negative control is 0.9% NaCl solution or water. 4.5 Cell activity detection
4.5.1 Only quantitative and effective detection methods can be used to determine cell activity, and the detection method for cell activity should be consistent with the detection method for three-dimensional tissue construction. The binding of non-specific dyes cannot interfere with the detection of cell activity. Dyes that can bind to proteins and those that cannot be metabolized (such as neutral red) are not suitable for this test. The most commonly used method is the MTT reduction method. The results of the MTT reduction method are accurate and reproducible, and other methods can also be used. 4.5.2 Place the skin sample in an MTT solution of appropriate concentration (such as 0.3mg/mL~1mg/mL), incubate at an appropriate temperature for 3h, then use a bath agent (isopropanol) to extract the blue methyl sulfone reduction product, and determine the concentration of methyl sulfone by measuring the D value in the wavelength range of 540nm~595nm.
4.5.3 If the test sample is not completely removed from the skin surface by washing, the chemical reaction between the test substance and the active dye may simulate the situation of cell metabolism, thereby leading to an erroneous judgment of cell brain activity. If the test substance directly acts on the active dye, a control group should be set up to detect and correct the interference of the test substance on the cell activity measurement results. 4.6 Interpretation of results
4.6.1 The relative cell activity of the negative control group is set to 100%, and each test sample obtains a 0D value. The percentage obtained by comparing this value with the negative control represents the relative cell activity. The critical percentage of cell activity used to distinguish corrosive from non-corrosive (or to distinguish between corrosives of different degrees) should be accurately recorded, or the statistical procedures used to evaluate the results and identify corrosives should be confirmed, and their rationality should be confirmed. Usually, these critical values are established during the optimization of the test method, confirmed in the pre-validation phase tests and formal validation studies. 4.6.2 If a relevant commercial model is used, the interpretation of the results of the prediction of chip corrosion shall refer to the instruction manual of the commercial model. 2
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5 Data and Reporting
5.1 Data
CB/T 27830—2011
All data results for each organization should be recorded in a table, such as: D value and calculated percentage of cell viability of the test substance, positive control and negative control, including data of repeated tests, average values and individual values. 5.2
2 Test report
The test report should include
a) Test substance and control group:
- Chemical name, such as International Union of Pure and Applied Chemistry (IUPAC nomenclature) or Chemical Abstracts (CAS name and CAS quotation marks (if known);
Purity and composition (mass fraction) of the chemical substance or preparation;- Plasticity properties, tree and this study
b) Justification for the skin model and experimental method used c) Test conditions:
Cell system used;
-- Calibration information of the instrument used to measure cell viability (e.g., spectrophotometer); Full supporting information for the skin model used in the mouse experiment, including validation Details of the experimental procedures
- Dosage of the test substance used;
Description of any modifications made to the experimental procedures; Historical references to the skin model;
Description of the evaluation criteria.
d) Results:
-- Data table for each test sample; . Description of other observations.
e) Discussion of results.
) Conclusion,
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A.1 The reference chemicals recommended by Chemicals
1,2-Diaminopropane
Acrylic acid
2-tert-butylphenol
Potassium hydroxide (10%)bzxZ.net
Sulfuric acid (10%)
Octoic acid
1-Amino-1:2,4-triazole
Eugenol
Benzyl bromide
Tetrazene
Isostearic acid (stearyl)
4-(Methyl)-benzaldehyde
Appendix A
(Informative)
Researched chemical substances
Table A.1 Researched chemical substances
Chemical Abstracts Service registration number
CA5-Na, 78-90-0
CAS-Na. 79-10-?
CAS-No. 88-18-6
CAS-Va, 1310-58-3
CAS-No, 7664-93-9
CAS No. 124-07-02
CAS-No. 584-13-4
CA5-No. 9 7-53-0
CAS-No. 103 63-9
CAS-No. 127-18-f
CAS-No.30399-84-9
CAS-No. 3446-89-7
Corrosive
Strongly corrosive
Strongly corrosive
Corrosive
Erosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Non-corrosive
Note: Most of the chemicals listed in the table are from the chemicals selected by the European Center for Validation of Alternative Methods (ECYAM) for international validation studies and are listed as follows: 1. The selection of chemicals is based on the following criteria: The number of corrosive and non-corrosive substances is equal. It covers most types of chemicals and is commercially available, including two types of materials: severely corrosive and weakly corrosive to distinguish the corrosion intensity. The selected chemicals should be based on laboratory management and have no other serious hazards except corrosiveness. Art examination documents
GB/T 27830--2011
[1- OECD(2001) Hamanised Integrated Classification System for Hurnan Health and Environ-mcntal Hazards nf Chemical Substances and Mixtures. OECD Serics on Testing and Assesscnt Num-ber 33. ENV/JM/MONO(2001)6,Paris http:// www, plis. oecd. org/olis/200ldoc. nsf/LinkTo/eny-jm-mono,2001,6
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[13] OECD(2002)Extended Expert Consultation Meeting on The In Vitro Skin Corrosion Test5
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Guideline Proposal, Berlin, lst-2nd November 200l, Secretariat's Final Summary Rcport,27th March20o2,OECD ENV/EHS,availeablc upon requcst front the Sccretariat[l4]Worth AP,Fentem JH,Balls M,Botham PA,Curren RD,Eerl LK,Esdaile DJ, Liebsch M(1998). An Evaluatian of the Prnposed OECD Testing Strategy for Skin Carrosion. ATLA 26:709-720[15] Mosmann, T. (1983).Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65: 55-63[16] Cannon, CL Neal, PJ, Southee.JA, Kubilus.J., and Klausnet, M. ,1994. New epi-dermal model far dermal iritancy testing. Toxic. in Vitro 8:889-891[17]Ponee,M.,Boelsma,E.,Weerheim,A.,Mulder,A.,Bouwstra,J., and Mommaas,M.2000, Lipid and ultrastructural characterization of reconstructed gkin models. International Journal ofPharmaccutic9.203.211-225
[l8] Tinois E,Gaetani Q,Gayraud B,Duppnt D, Rougier A,Pouradier Dx(1991). The Episkitnodel: Successful reconstruction of human epidcrmis in vitro. In In vitro Skin Toxicology, Edited by ARougier, AM Goidberg and HI Maibach: 133-140[1g] Tinois E, Tiollier J, Gaucherand M, Dumas H, Tardy M, Thivolet J(1ggl), In vitro anepost-transplantation differentiation of human keratinprytes grow on thc human typc IV collagen film of abilayered dertnal substitute.Exicrimental Ccll Resesrch193.310-319[20] Parenteau,NL,Bilbo,P ,, Molte,CJ,Mason,V, S.,and Rosenberg, H. (1992). Theorganotypic culture of human skin keratinorytes and fibroblasts to achieve form and function. Cyto-tcchnology 9:16:-t71
[21]Wilkins,LM,Watson,S,R,Prosky,SJ,Meunier,SF,Parenteau,NL(1994).Development of a bilayercd living akin construct for clinica) applications. Biotechnology and Bioengi-ncering 43/8:747-756
[22] Marshall,NJ,Goodwin,C, J.,Holt,SJ(1995). A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function,Growth Reguletian 5:69-84[23]Fentem,JH,Briggs,D.,Chesne,C.,Eltiot,G, R.,Harbell,JW,Heylings, JR,Portes,P ., Rouget, R. and van de Sandt,JJM,aud Botham,PA (20o1).A prevalidation study onin vitro tests for acute skin irritation: Iesults and evaluation by the Managcruent Team. Toxic. inVitro_15:57-93-t71
[21]Wilkins,LM,Watson,S,R,Prosky,SJ,Meunier,SF,Parenteau,NL(1994).Development of a bilayercd living akin construct for clinica) applications. Biotechnology and Bioengi -ncering 43/8:747-756
[22] Marshall, NJ, Goodwin, C, J., Holt, SJ (1995). A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function ,Growth Reguletian 5:69-84[23]Fentem,JH,Briggs,D.,Chesne,C.,Eltiot,G,R.,Harbell,JW,Heylings, JR,Portes,P.,Rouget,R. and van de Sandt,JJM,aud Botham,PA (20o1).A prevalidation study onin vitro tests for acute skin irritation: Iesults and evaluation by the Managcruent Team. Toxic. inVitro_15:57-93-t71
[21]Wilkins, LM, Watson, S, R, Prosky, SJ, Meunier, SF, Parenteau, NL (1994). Development of a bilayercd living akin construct for clinic) applications. Biotechnology and Bioengi -ncering 43/8:747-756
[22] Marshall, NJ, Goodwin, C, J., Holt, SJ (1995). A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function ,Growth Reguletian 5:69-84[23]Fentem,JH,Briggs,D.,Chesne,C.,Eltiot,G,R.,Harbell,JW,Heylings, JR,Portes,P.,Rouget,R. and van de Sandt,JJM,aud Botham,PA (20o1).A prevalidation study onin vitro tests for acute skin irritation: Iesults and evaluation by the Managcruent Team. Toxic. inVitro_15:57-93
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