GB/T 27825-2011 In vivo test method for skin absorption of chemicals GB/T27825-2011 Standard compression package decompression password: www.bzxz.net
This standard specifies the terms and definitions, test principles, test methods, test data and reports for in vivo test methods for skin absorption of chemicals. This standard is applicable to in vivo tests for percutaneous absorption of chemicals.
This standard was drafted in accordance with the rules given in GB/T1.1-2009.
This standard is consistent with the technical content of the Organization for Economic Cooperation and Development (OECD) Chemical Test Method No. 427 (2004) "Skin Absorption: In Vivo Test Method"
(English version).
This standard has been modified in the following structural and editorial aspects:
———A chapter on scope has been added;
———The "Introduction" and "Initial Considerations" in the original text of OECD 427 are used as the "Introduction" of this standard;
———The "Appendix" in the original text of OECD 427 is used as the content of "2 Terms and Definitions" of this standard;
———The measurement units are uniformly changed to the legal measurement units of China.
This standard is proposed and managed by the National Technical Committee for Standardization of Dangerous Chemicals Management (SAC/TC251). The
drafting units of this standard are: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Hubei Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, and China Chemical Economic and Technological Development Center.
The main drafters of this standard are: Liu Qingjun, Li Chaolin, Lin Zheng, Zeng Xiandong, Yang Ting, Zhang Jianfeng, Guo Mingxing, Chen Jianjun, Zhao Hui, Feng Hanli, and Wang Zhenhua.

ICS13,300;11.100
National Standard of the People's Republic of China
GB/T 27825--2011wwW.bzxz.Net
Chemicals
Skin absorption
In vivo test method
Chemicals-Test method for skin absorption-In viva2011-12-30 Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Administration of Standardization of the People's Republic of China
Implementation on August 1, 2012
This standard was drafted in accordance with the rules given in GB/T1.1-2009. CB/T 27B25-2011
This standard is consistent with the technical content of the Organization for Economic Cooperation and Development (OECD) Chemical Test Method No. 127 (2004) & Skin absorption: In vivo test method (English version).
This standard has been modified in the following structural and editorial aspects: 1. Scope--Chapter added;
1. The "Introduction" and "Initial Considerations" in the original text of OECD 42? are used as the "Introduction" of this standard; 2. The "Appendix" in the original text of OECD 427 is used as the content of "2 Terms and Definitions" of this standard; 3. The measurement units are uniformly changed to the legal measurement units of my country. This standard is proposed and managed by the National Technical Committee for Standardization of Dangerous Chemicals Management (SAC/TC 251). The drafting units of this standard are: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Beishan Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, and China Chemical Economic and Technological Development Center. The main drafters of this standard are: Liu Qingjun, Li Chaolin, Lin Zheng, Zeng Xiandong, Yang Ting, Zhang Jianfeng, Guo Ming, Chen Jianjun, Zhao, Feng Hanli, Wang Zhenhua.
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GB/T 27825-2011
The technical content of this standard mainly refers to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Methods Guide No. 427 (2004), which provides a set of in vivo test methods for chemical skin absorption testing. Human exposure to chemicals is mainly through the skin, and most current toxicology studies use experimental animals for oral exposure. The in vivo test method for percutaneous absorption described in this guide provides an inevitable link for the safety assessment of chemical percutaneous exposure extrapolated from oral tests.
Chemical substances need to penetrate It takes multiple layers of skin cells to reach the circulatory system. For most substances, the dead cells of the skin's stratum corneum determine the rate at which they penetrate the skin. The permeability of chemicals to the skin also depends on factors such as the lipophilicity of the chemical itself, the thickness of the outer layer of the epidermis, and the relative molecular mass and concentration of the substance. In general, the skin of rats and rabbits is more permeable than human skin, while the skin permeability of guinea pigs, pigs, and monkeys is more similar to that of humans. There are two methods for testing the percutaneous absorption of chemicals, in vivo and in vitro. The in vivo test method can provide good information on percutaneous absorption of experimental animals of different species. In vitro test methods have also made progress over the past few years. In vitro test methods, in which the chemical substance is primarily tested through the full or partial thickness of animal or human skin to reach the intended fluid reservoir, are described separately in the OECD Guide to Test Methods. In certain cases, the OECD Guide to Skin Absorption Studies 2 can be used as a reference for selecting an appropriate test method. This document provides more details on the applicability of in vivo and in vitro test methods. The in vivo test methods described in this guide can determine the penetration of the test substance through the skin into various parts of the body. This method has been widely used for many years: $. Although in vitro percutaneous absorption studies are applicable in many cases, there are some situations where only in vivo test studies can provide the necessary data. The advantages of in vivo test methods are that they use physiologically and metabolically intact systems, use a variety of experimental animal species that are commonly used in dermal studies, and can be applied to other species with appropriate modifications. Of course, this method also has its disadvantages: the use of live animals, the need to use isotope-labeled materials to obtain reliable results, the difficulty in determining the early stages of percutaneous absorption, and the differences in skin permeability between the preferred species (rat) and humans. "- In general, animal skin permeability is better, so the test may overestimate the percutaneous absorption in humans. In addition, corrosive substances such as acids and alkalis are not tested on live animals.
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1 Scope
In vivo test methods for skin absorption of chemicals
GB/T27825--2011
This standard specifies the terms and definitions, test principles, test methods, test data and reports for in vivo test methods for skin absorption of chemicals. This standard applies to in vivo tests for percutaneous absorption of chemicals. 2 Terms and definitions
The following terms and definitions apply to this document. 2.1
Unabsorbed dose
absorbeddose
The amount of the test substance washed off the skin surface and on the protective device after poisoning, including the amount volatilized from the skin surface during poisoning.
Absorbed dose (in vivo) absorbed dose (in vivo) In vivo) after the test substance is removed from the skin of the test site, including urine, faeces, exhaled air (if tested), blood, tissue (if collected), and test substances retained in the corpse. 2.3
Absorbable dose
The maximum amount of test substance remaining on the skin surface or in the skin tissue after washing. 3 Experimental principles
The test substance (preferably labeled with an isotope) is applied to the hairless skin of the animal, and one or more representative and appropriate groups are set up for the test. During the predetermined exposure time, the skin is exposed to the test preparation under the cover of an appropriate (non-closed, semi-closed or closed) shielding device. Before, during and after the exposure, the test animals are kept in separate metabolic cages, and the animal excreta and exhaled air are collected. At the end of the exposure period, the shielding device is removed and the skin is washed with an appropriate detergent, and the shielding device and washing fluid are retained for analysis. . Usually, several animal groups are set up in each dose group; one group is killed at the end of the exposure period, and the other groups are killed in turn after a predetermined time interval. The remaining animals are killed at the end of sampling, blood is collected, the skin of the test site is removed for analysis, and the body is analyzed to determine the amount of the test substance that has not been excreted. The collected samples are analyzed by appropriate methods and the degree of percutaneous absorption of the test substance is evaluated =3.-51. 4 Test methods
4.1 Animal selection
Rats are the most commonly used species, and hairless series strains with skin absorption rates more similar to humans can also be used. Male healthy adult animals of commonly used laboratory strains are generally selected. At the beginning of the test, the difference in animal weight does not exceed 20% of the average weight. For example, the appropriate weight of male rats is 200g~~250g, which is in the upper half of this range. 1
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GB/T 27825--2011
4.2 Number and sex of animals
Generally, a test group consists of at least four animals of a single sex. A group of animals is killed at each predetermined time point in the test, such as at the end of exposure (usually 6h or 24h) and at subsequent time points (such as 48h, ?2h). If there are data showing that there is a significant difference in the transdermal toxicity between male and female animals, the more sensitive sex should be selected, otherwise one sex can be selected. 4.3 Rearing environment
The room temperature of the animal laboratory should be 22℃±3℃, and the relative humidity should generally be 50%-60%, at least 30%, and preferably not more than 70% (except during cleaning). Artificial light is used, with 12h light/dark alternation. Regular feed is used for feeding, with sufficient supply and free drinking water. During the test (including the adaptation period before the test), animals should be raised individually in their own metabolic cages, and try to avoid food and water overflowing from the container to affect the test results.
4.4 Animal preparation
Before the test begins, each animal should be marked to distinguish it and kept in separate cages for at least 5 days to adapt to the laboratory environment. Then, about 24 days before exposure, remove the hair on the shoulders and a certain area of ​​the back of each animal, and gently wipe the hairless skin surface with propylene glycol to remove grease. Soap residue may promote the absorption of the test substance, so washing with soapy water is not recommended. The permeability of broken skin is different from that of intact skin, and care should be taken to avoid damaging the skin. The test area should be large enough to ensure that the amount of skin absorption per square centimeter can be reliably calculated, preferably not less than 10 cm. This area is suitable for rats weighing 200g to 250g. The prepared animals are returned to the cage for use. 4.5 Test substance
The test substance is the substance used to study its permeability characteristics. The test substance is preferably radioactively labeled. 4.6 Preparation of test substances
Test preparations (including pure products, diluted preparations containing test substances or preparations directly applied to the skin, etc.) should be consistent with the possible exposure of humans or other potential target species (or be ideal substitutes). Differences in the preparation process should be reasonably corrected. If necessary, the test substance can be dissolved or suspended in an appropriate excipient. For non-aqueous preparations, their absorption characteristics and potential reactivity with the test substance should be understood.
4. 7 Skin toxicity
Use a known amount of the test preparation evenly on a test site with a specific area on the skin surface. The most common amount used is the amount that humans may contact. For solid test substances, it is usually 1tug/cm~5mg/cm, and for liquid test substances, it can be up to 10μL/cm. Other amounts can be used according to the expected use, research purpose or physical properties of the test preparation, but there should be appropriate reasons. After toxicity, the test site should be avoided from touching. Typically, the test site should be covered with a non-occlusive covering device (e.g., permeable nylon gauze). An example of a typical device is shown in Figure 1. In some undetermined cases, the test site should be covered with an occlusive covering device. If the volatilization of semi-volatile substances causes the recovery rate of the test substance to exceed the acceptable range (see also 4.10), the test device should be covered with an activated carbon filter to capture the shed test substance (see Figure 1). Any device must not damage the skin, nor absorb or react with the test preparation. After exposure, the animals are returned to individual metabolic cages for collection of excreta.
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GB/T 27825—2011
Active filter
Or gauze observation
Spiral spot phase
Thread disease
Chlorophenylpropionic acid
Salt adhesive stripping
--skin peptide
Figure 1 A typical quick cover device design example for covering and protecting the skin of the test site Figure 4.B Beam poisoning time and sampling
The poisoning period refers to the time interval between the beginning of skin contact with the test substance and the removal of the test substance by washing. The poisoning time (usually 6 h or 24 h) should correspond to the normal possible human contact time. After poisoning, the animals are kept in the metabolic cage until the predetermined time of sacrifice. At the end of poisoning, the test skin is observed and any visible irritation symptoms are recorded. Metabolic cages should be used to collect urine and feces separately throughout the study and to collect and quantify 1C-labeled carbon dioxide and volatile 1C compounds (greater than 5%) when they are produced. Fluids, feces, and excreta (e.g., 1C-labeled carbon dioxide and volatile 1C compounds) should be collected separately from each group at each sampling time point. Open cages may be used if there is sufficient information to demonstrate that little or no radioactive metabolites are produced. Animals should be observed regularly for toxicity or abnormal reactions throughout the study. During the exposure period, feces should be collected from the first 24 hours after skin fusion and daily thereafter until the end of the study. In general, three fecal collection intervals are sufficient, but the characteristics of some test preparations or existing kinetic data may require more collection time points. At the end of the exposure period, the cover is removed from each animal and retained individually for analysis. The test site skin of all animals should be cleaned at least three times with a swab and a cleaning agent, taking care to avoid contamination of other sites. The cleaning agent should be a representative common hygienic cleaning agent such as soap and water: Finally, dry the skin, cover the test site skin with a new clean cover plate, and return these animals to separate metabolic cages to form the experimental animal group at the next time point. All scrubs and washing solutions should be saved for analysis. 4.9 Animal Euthanasia
Each animal in each experimental group should be euthanized at the specified time point, blood samples should be collected, and protective devices or covers should be removed and analyzed. Cut the test site skin of each animal and the corresponding blank control site skin for separate analysis tests. The test site skin can be separated from the stratum corneum and the underlying dermis, which can provide more information on the distribution of the test substance. The distribution of the test substance in this period after infection will provide some indication of its transport in the stratum corneum. After the final cleaning of the skin and euthanasia of the animals, the protective cover should be removed to facilitate the stratification of the skin. Cut the skin of the test area from the body of the test rat in a circular shape and pin it to a board. Stick the adhesive strip to the skin surface and press lightly. The strip will stick part of the stratum corneum. Continue to stick the strip repeatedly until the strip no longer adheres to the skin surface, indicating that the stratum corneum has been completely removed. Combine all the strips from the same animal into a separate container and add tissue digestion fluid to dissolve the stratum corneum.
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Retain the body of each animal for experimental analysis. Before conducting an absorption dose test on the animal body, each possible target tissue can be separated and tested separately. Generally speaking, only the total content of the body is analyzed. If there are other research suggestions, the target tissue can also be separated and tested separately. When the animal is euthanized, the urine in the bladder should be combined with the previously collected urine. In addition, after collecting feces in the metabolic cage, the cage and capture device should be rinsed with an appropriate solvent and collected, and other potentially contaminated instruments should be treated similarly.
4.10 Analysis
All tests should achieve adequate recovery (e.g., average 100% ± 10% efficacy), and reasons should be given for exceeding the recovery range. The dose used for each sample should be analyzed using an appropriate and valid method. Statistical analysis should take into account the deviation between the replicates of each dose group. 5 Test Data and Report 5.1 Test Data The following measurements of the test substance and/or metabolite should be made for each animal at each sampling time point (in addition to the data of individual animals) in order to calculate the absorbed dose, unabsorbed dose and absorbable dose, and the data at different sampling time points should be reported as average values ​​by group: - amount on protective gear; - amount cleared from the skin; - amount on/in the skin and cannot be washed off the skin; - amount in blood samples; - amount in feces and exhaled breath (if applicable); - amount retained in several organs tested separately under similar conditions. The maximum amount of the test substance and/or metabolite in feces, exhaled breath, blood and body samples is used to determine the total amount absorbed at each time point, thereby calculating the amount of the test substance absorbed per square centimeter of skin after exposure. 5.2 Test Report
The test report should meet the requirements specified in the test protocol, including the rationality of the test system used, which includes the following aspects: b) Test substance:
Identification data [e.g., Chemical Abstracts Service (CAS) number, source, purity (radiochemical purity), known impurities, catalog number1;
physical properties, physicochemical properties (e.g., pH, volatility, solubility, stability, molecular weight and igP). b) Preparation of test substance:
|Formulation and reasons for use;
Detailed process of preparation of test substance, amount used, concentration achieved, excipients, stability and homogeneity. c) Test animals:
Species/strain used;
|Effective dose, age and sex of animals:
: · Source of animals, breeding environment, feed, etc.;
Weight of each animal at the beginning of the test. d) Test conditions:
GB/T 27825—2011
- Detailed process of poisoning (test site, analytical method, closed/non-closed, volume, extraction, inspection), detailed description of food and water quality.
e) Test results:
- What are the toxic symptoms;
- List of absorption data (expressed in rate, number and percentage): overall recovery rate of the test;
- Interpretation of the results, compared with the existing data on the percutaneous absorption of the test substance. f) Discussion of the results.
Conclusion. OECD, Paris[3] ECETOC(1993), Percutanrous Absorption. Fur opean Cenlre for Erotoxicplogy and Toxi-cologyofChemicals, MonographNo.20[4] Zendzian RP(1989). Skin Penetration Method suggested for Environmental ProtectionAgency Requirements. J. Am. Coll. Toxicol. .829-835[5] Krmppainen BW, Reifenrath WG(1990). Methods far skin absorption. CRC Press BocaRaton,FL,USA
[6] EPA(1992). Dermal Expasure Asseyment: Principles and Applicationa. Exposure Assess-ment Group, Office of Hcelth and Environmental Aasessment[7] EPA(1998).Health Effects Test Guidelines ,OPPTS 870-7600,Dermal Penetration.Office of Prevention,Pesticides,and Toxic Substances[8] Bronaugh RL, Wester RC,Bucks D),Maibach HI and Sarason R
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