Some standard content:
ICS13.300,11.100
National Standard of the People's Republic of China
GB/T27818—2011
Chemicals
Skin absorption
In vitro test method
Chemicals-Testing method for skin absorption--In vitroIssued on December 30, 2011
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 August 1, 2012
This standard was drafted in accordance with the rules given in GB/T1.1--2009. GB/T278t8—2011
This standard is consistent with the technical content of the Organization for Economic Cooperation and Development (OECD) Chemical Test Method No. 428 (2004) "Skin Absorption: In Vitro Test" (British version).
This standard has made the following structural and editorial changes: added the scope chapter;
added OECD428 The "Initial Consideration\ and\ Introduction" in the original text shall be used as the introduction of this standard; the "End of Procedure\ and Analysis\" in the original text of ECD428 shall be used as "4.10 Dyeing Time and Sampling" of this standard; - The "Data and Report\ and "Conclusion" in the original text of ECD428 shall be used as "5 Test Data and Report" of this standard; - The "Definition" in the original appendix of OECDM28 shall be used as "2 Terms and Definitions" of this standard; - The measurement units shall be uniformly changed to the legal measurement units of my country. This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous 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, Tianjin Center for Disease Control and Prevention, and China Chemical Economic and Technological Development Center.
The main drafters of this standard are: Yu Yanrang, Liu Qingjun, Lin Zheng, Yang Deyi, Liu Baofeng, Liu Jing, and Zhang Ming.TTTKONYKAA
GB/T 27818—2011
This standard refers to OECD Chemical Test Method No. 428 (2004) Skin Absorption: In Vitro Tests (English version). This guideline provides methods for in vitro skin absorption tests, which can be used in conjunction with OECD skin absorption (in vivo tests) or alone. This test method recommends the OECD research document on skin absorption as a guidance document to assist in the design of experiments based on this method: the OECD guidance document will help to select appropriate in vitro operating procedures in special circumstances and ensure the reliability of the results obtained by this method.
Methods for detecting skin absorption and transdermal absorption can be divided into two categories: in vivo tests and in vitro tests. Methods for in vivo skin absorption have been established and provide information on the pharmacokinetic metabolism of different species of animals. In vivo test methods will be developed in another OECD document. In vitro methods for measuring skin absorption have been used for many years. Although formally validated in vitro absorption studies using this method have not been conducted, OECD experts reached a consensus in 1999 that there are sufficient data to support this method [], and this is supported by the detailed information provided by the method, including important data from direct comparisons of in vitro and in vivo test methods. A number of monographs have discussed this view and provided detailed information on the use of in vitro test methods-21. In vitro tests can measure the diffusion of chemicals into body fluids through the skin, and can measure this diffusion using inactive skin alone, or can use fresh, metabolically active skin to measure both this penetration and skin metabolism. This method has particular use in screening and comparing different chemicals that enter or are transported through the skin in different ways, and in providing a useful model for evaluating human percutaneous absorption. In vitro tests are not suitable for all situations and types of chemicals. They are only suitable for initial qualitative evaluation of skin penetration, and in some cases, further reference should be made to in vivo test data. OECD Chemical Test Method No. 428 Skin Absorption: In Vitro Tests (2004) (English version) should further clarify the applicability of in vitro test methods and provide additional details to support these conclusions in the OECD Expert Meeting Report.
OECD Chemical Test Method No. 428 Skin Absorption: In Vitro Tests (2001) (English version) proposes the general principle of using ex vivo skin to measure the percutaneous absorption and transport of test substances. Ex vivo skin can be obtained from a variety of mammals, including human skin. Because the permeation layer is composed of the inactive stratum corneum, the permeability of the skin is retained after ex vivo: the mechanism of active transport of chemicals through the skin has not been confirmed. Studies have shown that the skin has the ability to metabolize some chemicals during percutaneous absorption [5]. Although this will affect the properties of the chemicals entering the blood, this process is not the limiting step for the actual absorbed dose. TTTKANTKACA
1 Scope
In vitro test methods for skin absorption of chemicals
GB/T27818—2011
This standard specifies the terms and definitions, test principles, test methods, test data and reports for in vitro test methods for skin absorption of chemicals. This standard is not applicable to all situations and all types of chemicals for percutaneous absorption, but only to the initial evaluation of skin penetration. In some cases, further reference should be made to in vivo test data. 2 Terms and definitions
The following terms and definitions apply to this document. 2. f
Unabsorbed dose
tunabsorbeddose
The amount of the test substance washed off the skin surface and on the cover device after poisoning, including the amount volatilized from the skin surface during the poisoning process.
Absorbed dose (in vitro test) absorbed dase: in vitro The amount of the test substance reaching the receiving fluid or circulatory system within a certain period of time. 2.3
Absorbable dose (in vitro test) The amount of the test substance remaining on the skin surface or in the skin tissue after washing. 3 Test principle
The test substance (which may be a radiolabeled substance) acts on the skin surface that divides the diffusion cell into two parts. Under certain conditions, the chemical needs to act on the skin for a certain period of time, and then use an appropriate cleaning method to remove the chemical from the skin surface. During the whole test, the receiving fluid is collected at different time points and analyzed for the test substance and metabolites. If a metabolic activation system is used, the metabolites of the test substance should be analyzed by appropriate methods. At the end of the test, the distribution of the test substance and its metabolites is measured under appropriate circumstances. Under appropriate conditions described in this standard and guidance documents, the absorption of the test substance over a certain period of time is monitored by analyzing the receiving fluid and the test skin. Unless it can be proved that the test substance is absorbed only by the absorption fluid, the amount of the test substance remaining in the skin should be considered as the amount absorbed. In order to further analyze the data, other components (those washed off from the skin surface and those remaining in the skin) should also be analyzed, including the distribution and recovery of all test substances. In order to demonstrate the accuracy and stability of the test system, the test results of the relevant chemicals should be consistent with the conclusions of the literature published using this method. This requirement should be achieved by testing appropriate reference materials (preferably with a lipophilicity similar to that of the test substance), or by providing sufficient empirical data for a variety of reference materials with different lipophilicity (such as caffeine, malic acid, testosterone). 4 Test method
4.1 Diffusion cell
The diffusion cell is divided by the skin and includes a supply cell and a receiving cell (Figure 1 is a typical diffusion cell sample). The diffusion cell should have a good seal around the skin to facilitate sampling and good mixing with the receiving solution in contact with the underlying skin. And the temperature of the diffusion cell and its contents can be well controlled. Both static osmotic and circulating diffusion cells can be used. Under normal circumstances, the supply chamber is kept open during the exposure to the finite dose test substance. However, when using the infinite and partially finite dose test substance, the supply chamber should be closed. (Automatic) Sampler
Set the sampling time
Active sample hood
(For volatile samples?
Supply cell
Skin waist (2.54 cm)-
Support tray
Stirring bar
(Automatic) Pipette/inject sample into liquid
Improve counting effector or high pressure liquid chromatograph
Standard sample rent
By replacing fresh receiving wave body to maintain a fixed volume
Water-filled control receiving range/receiving temperature, maintained at 32℃±1℃
Figure 1 Example of a typical static diffusion cell for in vitro percutaneous absorption test 4.2 Receiving solution
Although other solvents have been shown to be acceptable as receiving solutions, physiologically beneficial solutions should be preferred. The precise composition of the receiving solution should be provided, and it should be confirmed that the test substance is fully soluble in the receiving solution and does not affect skin absorption; in addition, the receiving solution should not affect the integrity of the skin. In a circulating flow system, the flow rate should not prevent the test substance from diffusing into the receiving solution. In a static osmotic system, the receiving solution should be continuously stirred and sampled regularly. If the metabolic effects on the skin are to be studied, the receiving solution must be able to maintain the activity of the skin during the entire test.
4.3 Skin preparation
Specimens taken from human or animal skin can be used. Human skin specimens may comply with national and international ethics committee conventions (13). Living skin is preferred, but inactive skin specimens that have been shown to be intact may also be used. The epidermis (enzymatically, thermally or chemically separated) or thick skin (typically 200 μm to 400 μm thick) separated with a dermatome can be selected. However, the use of very thick skin (approximately 1 mm) should be avoided unless the chemical is specific and the entire skin layer is required for testing. The skin type, anatomical location and skin preparation technique should be determined. At least four replicates are required for each test sample. 4.4 Skin integrity wwW.bzxz.Net
Skin specimens should be properly prepared. Improper handling may damage the stratum corneum, so the integrity of the skin specimen should be checked. When the metabolic function of the skin is to be studied, the isolated fresh skin should be used as soon as possible and its metabolic activity should be maintained under known conditions. In general, fresh ex vivo skin should be used within 24 h. Use within, but the appropriate storage period may vary depending on the metabolic enzyme system and storage concentration. If the skin specimen has been stored before use, it is necessary to prove that its barrier function remains intact. 2
TTIKANYKACA
4.5 Test substance
The test substance refers to the substance used to study its penetration characteristics. The test substance is preferably radioactively labeled. 4.6 Test substance preparation
CB/T27818—2011
The test preparation (including pure product, diluted preparation or preparation containing the test substance applied directly to the skin, etc.) should be consistent with the possible exposure of humans or other potential target species (or an ideal substitute). Differences in the preparation process should be reasonably corrected. 4.7 Test substance concentration and composition
Usually, there is more than one test substance concentration, which should cover the possible human infection range. Similarly, the concentration range of typical components should also be considered.
4. 8 Skin exposure
In general, the dose of chemicals that humans are exposed to is usually limited. Therefore, the agent used to simulate human exposure usually uses 1 mg to 5 mg of solid chemicals or 10 μL of liquid chemicals per square meter of skin surface. The dosage is adjusted according to the study subjects, the physical characteristics of the test sample and the specific conditions of use. For example, when a large dose of chemical is used per unit area, the amount of chemical contacting the skin surface may be unlimited.
4.9 Temperature
The passive absorption of chemicals is affected by temperature. The diffusion chamber and the skin should be kept constant and close to the normal temperature of the skin (32 ± 1). Different diffusion chambers require different water baths or heating to a certain temperature to ensure that the receptor/skin temperature is in its positive physiological range. The relative humidity is preferably between 30% and 70%.
4.10 Exposure time and sampling
It is possible to expose the skin to the poison throughout the test process, or it can be exposed for a shorter period of time (such as a specific model simulating human exposure). Excess test substance is washed off with appropriate cleaning agents and then collected and washed for analysis. The cleaning procedures for the test substance depend on the conditions of use of the test substance and should be justified for the cleaning procedures for the test substance. It is usually required to take into account the characteristics of full absorption and to sample for 24 hours. Since skin integrity deteriorates over 24 hours, sampling should not be performed for more than 24 hours. For test substances that penetrate the skin rapidly, exposure to 24 hours may not be necessary, but for low-penetration test substances, longer exposure times may be required. The sampling frequency of the receiving fluid should be based on the absorption characteristics of the test substance, and the sampling frequency should be given in a graphical form. 4.11 Results and Analysis
All components of the test system should be analyzed and the recovery determined, including the supply reservoir, skin surface wash, prepared skin, and receiving fluid/receiver reservoir. In some cases, the skin should be separated into two parts: the exposed skin and the skin under the flange of the receiving reservoir, thereby dividing the stratum corneum, epidermis, and dermis into separate parts for analysis. Adequate recovery is required for all these test substances (the mean of the effective activity should be 100 ± 10%, and any deviation should be explained). Appropriate technical methods should be used to analyze the amount of test substance in the receiving fluid, skin, skin surface wash, and instrument wash.
5 Test data and report
5.1 Data
The analysis results of the receiving solution, the distribution and absorption of the test substance in the test system at different times should be listed. For limited toxicants, the amount of chemical washed off the skin, the amount bound to the skin (analysis of different skin layers) and the amount of test substance in the receiving solution (ratio, amount and percentage of the used agent) should be calculated. The results of the percutaneous absorption test can sometimes only be expressed by the data of the receiving solution. However, when the test substance still remains in the skin at the end of the test, the total absorption should include the amount remaining in the skin. For infinite dose exposure, the permeation coefficient (K,) needs to be calculated, but the absorption percentage is meaningless. 5. 2 Test report
The test report should include the requirements specified in the protocol. In addition to the proof of the application of the test system, it should also include the following: 5.2.1 Test substance:
Physical characteristics, physicochemical properties (at least including relative molecular mass and IgP distribution ratio), purity (radiochemical purity); identification information (such as batch number;
Solubility in the receiving solution.
Preparation of test substance:
a) Components and principles of the preparation used;
b) Homogeneity.
5.2.3 Test conditions:
Skin source and location, preparation method, storage conditions before use, any pretreatment (cleaning, antibiotic treatment, etc.), skin integrity measurement, metabolic status, reason for use; design of diffusion cell, composition of receiving fluid, flow rate of receiving fluid or sampling frequency and procedure; b)
Details of the test agent used and the basis for dose selection: duration of exposure:
Details of removal of the test agent from the skin, such as skin washing: details of the skin analysis and separation technology used to illustrate skin distribution! Diffusion cell and instrument cleaning procedures 5
Detection method, extraction technique, detection limit and validity of the analytical method. 5.2.4 Results:
Recovery of the test substance (dose used = skin washout + amount in the skin + receiving solution | diffusion cell cleaning solution); recovery of each reaction cell;
absorption of the test substance,
percutaneous absorption data (expressed as penetration rate, penetration amount or percentage) 5.2.5 Discussion of results.
5.2. 6 Conclusion.
References
GB/T 27818-—2011
[17 OECD (2004), Test Guidelinc 427,Skin absorption;in uivo Method. OECD,Paris[2_OECD (2004). Guideline Document for the Conduct of Skin Absorption Studies. OECD, Paris[3] OECD (2000), Rcporl ol the Meeting of the OECD Extended Steering Committee for Percu,taneous Absorption Testing:Annex 1 to ENV/JM/TG(2000)5.OECD,Paris[4 Kemppainen BW and Reifenrath WG. (199o). Methods for skin absorption. CRC Press:BocaRaton
[5?Franaugh RL and Collier SW. (1991). Absorptian Studies,in Irvitro Percutatteous Ahserption Principles, Furdamertalr and Applications ,RL Bronaugh and HI Maibach,Eds. CRC Prcss,Hoca Ratonl,237-241[6] Bronaugh RL and Maibech HI, (19gl). In vitro Perecutaneuus Ahserpton:Principtes,Fun-damentais and Applications ,CRC Press,Boca Retan[?] European Center for Ecotoxicology and Toxicology of Chemicals (1g93), Monograph No20,Perrutaneous Absorption,ECETOC,Brussels[8] Diembeck W.Beck H,Benech-Kieffer F,Courtelleruont P,Dupuis J,Lovell W,Paye M,Spengler J,Stciling W (1999). Test Guidelines for In vitro Assessment of Ierrnal Absorption and PercutancousPenetration of Cosmctic Iugredients.Fd Chem Tox,37,191-205[9]Recommended Protocol for I uitro Pcrcutaneous Absurption Rate Studies (1996),USFederal Rcgistcr,Vol, 61,N. 65[10] Howes D,Guy R, Hadgrelt J. Heylings JR et at. (1996). Methods for assessing percutaneous ab-sorption, ECVAM Workshop Report ATLA 24.8l R10E11] Schaefer H and Redelmeier TE (1996). Skin barrier: principles of pcreutaneous absorptionKarger, Basel
[127Roberts MS and WaltersKA,(1998).Dermal absorption and toxicity assessment.MarcelDckker,New York
[13WMA (2008). WMA Declaration of Helsinki-Ethical Principles for Medical Research InvolvingHurnan Subjects.59th WMA General Assembly,Seoul[14] Jewell,C., Heylings,JR.,Clowcs,HM. And Williarns, FM (2000). Pcrcutaneous absorption andmetabolism of dinitrochlorobenzene in vitro. Arch Toxicol 74:356-365
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.