Come Clean: Skin Irritation and Corrosion Tests Resources for Companies
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There are multiple reasons to transition your company from using in vivo methods to in vitro, or to require your ingredient suppliers to conduct in vitro testing. In 2002, the Organisation for Economic Co-operation and Development (OECD) adopted a tiered approach for dermal irritation and corrosion testing as described in the revised test guideline TG 404.
The aforementioned test guideline states that “in the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all available data relevant to the potential dermal corrosivity/irritation of the substance have been evaluated in a weight-of-the-evidence analysis.” A weight-of-evidence approach requires that all available information be gathered before conducing testing.1,2 This includes both human and animal data, physicochemical properties, data on analogous substances, and eye irritation data. The chart below, taken from Macfarlane et al.3 gives an example of how this evidence, and in vitro tests, can be combined to accomplish skin testing without animals.
Many validated nonanimal methods are available for assessing the skin irritation and corrosion potential of cosmetic products and ingredients. It is possible to assess both irritation and corrosion endpoints with a human skin model that consists of human-derived epidermal keratinocytes (the predominant cell type in the epidermis) cultured to form a model that retains the characteristics of human tissue, termed a Reconstructed Human Epidermis (RhE) model. The use of these models is outlined in OECD test guidelines 431 In Vitro Skin Corrosion: Human Skin Model Test and 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Another in vitro OECD test guideline, 435 In Vitro Membrane Barrier Test Method for Skin Corrosion, is also available for skin corrosion testing. Each model has its own “applicability domain” of test substances for which they are best suited.
To assess the potential for skin irritation, five RhE models exist and are available for commercial use: EpiDerm, SkinEthic, CellSystem EST1000, LabCyte EPIMODEL-24, and EpiSkin. In-depth information on the development and validation of three of these models, which can be used in OECD test guideline 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, is available here. The remaining two have been validated and will be added to the test guidelines shortly. Additionally, evaluating skin irritation using these in vitro models “are said to be cheaper”4 and are faster than conducting tests on rabbits – a rabbit test takes 14 days and an in vitro test takes four days.
To evaluate the risk of skin corrosion we recommend using human-based models. OECD test guideline 431 In Vitro Skin Corrosion: Human Skin Model Test. EpiDerm, SkinEthic, EpiSkin, and CellSystem EST1000 are all validated RhE models that can be used to conduct this test guideline. OECD test guideline 435 In Vitro Membrane Barrier Test Method for Skin Corrosion is a physico-chemical test that assesses the time it takes for a chemical to break through a synthetic macromolecular membrane. Corrositex is an available model that assesses skin corrosion in this way.
An in vitro skin irritation test using the EpiDerm RhE can been seen here. This 21-minute video details the four-day procedure. As is noted in the abstract of the video, in vitro methods for assessing skin irritation and corrosion are vital for marketing products in the European Union (EU). Under the EU Cosmetics Directive, in vivo methods for testing skin irritation and corrosion cannot be conducted for both final products and ingredients anywhere in the world.5
Many of the models mentioned above are available for purchase from tissue suppliers. Follow the links connected to the names of the models for contact information. Companies can also take advantage of testing services that specialize in in vitro testing, such as CeeTox or the Institute for In Vitro Sciences.
If your company or ingredient supplier is interested in transitioning to in vitro methods please contact PCRM to speak with one our experts.
OECD Test Guideline 431 In Vitro Skin Corrosion: Human Skin Model Test
OECD Test Guideline 435 In Vitro Membrane Barrier Test Method for Skin Corrosion
OECD Test Guideline 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Explanatory Background Document to the OECD Test Guideline On In Vitro Skin Irritation Testing
Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations6
Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labeling and Pack- aging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.
A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Skin irritation7
Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkinTM, EpiDermTM and SkinEthicTM reconstructed human epidermal equivalents. For skin irritation, EpiSkinTM, EpiDermTM and SkinEthicTM are validated as stand- alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.
Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data8
Background: Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated.
Objective: A key difficulty in confirming the validity of in vitro methods is that animal data are scarce and of limited utility for prediction of human effects, which adversely impacts their acceptance. This study examines whether in vivo or in vitro data most accurately predicted human effects.
Methods: Using the 4-hr human patch test (HPT) we examined a number of chemicals whose EU classification of skin irritancy is known to be borderline, or where in vitro methods provided conflicting results.
Results: Of the 16 chemicals classified as irritants in the rabbit, only five substances were found to be significantly irritating to human skin. Concordance of the rabbit test with the 4-hr HPT was only 56%, whereas concordance of human epidermis models with human data was 76% (EpiDerm) and 70% (EPISKIN).
Conclusions: The results confirm observations that rabbits over predict skin effects in humans. Therefore, when validating in vitro methods, all available information, including human data, should be taken into account before making conclusions about their predictive capacity.
In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy Results and performances with 184 cosmetic ingredients9
Following considerable interest in the development of in vitro model substitutes, reconstructed human skin or epidermis available as standardized kits, allow the measurement of parameters linked to the safety of topically applied products. Able to mimic in vivo situation, the EPISKINTM 3-D model (SkinEthic Laboratories) was involved in the formal ECVAM sponsored acute skin irritation validation. EPISKINTM 0.38 cm2 was validated as a full replacement method by the ESAC (April 2007 statement).
The protocol was based on a short treatment time (15 min) followed by an extended 42 hour post- treatment incubation period. The prediction model mainly based on the validated viability measurement (MTT) combined with the useful adjunct IL-1α and appropriate cut-off values allowed the drawing up of 2 chemical classes: Irritants (EU risk, R38) and non-irritants (EU risk, no classification). Applied to a set of 184 raw ingredients covering diverse physical-chemical categories, sensitivity, specificity and accuracy of the prediction model were 85%, 86% and 86% respectively. Results demonstrated the usefulness of the tiered strategy combining two complementary end points MTT + IL-1α as a decision-making tool for skin irritancy hazard identification. The present work is part of a strategy assessing performances on a large number of chemical realistically relevant to industrial needs in order to concretely determine applicability domains while understanding the remaining gaps.
1 Eskes, C., 2010a. Guidance document on the application of alternative methods in the Regulatory Assessment of Chemical Safety Related to Human Skin Corrosion and Irritation: current status and future prospects. FOPH, pp. 65. Website accessed on June 18, 2012 at: http://www.orange--house.eu/assets/r%20rt(2009)%203%201%20final.pdf.
2 Eskes, C., 2010b. Guidance document on the application of alternative methods in the Regulatory Assessment of Chemical Safety Related to Human Eye Irritation and Severe Irritation: current status and future prospects. FOPH, pp. 65. Website accessed on June 18, 2012 at: http://www.orange--house.eu/assets/r%20rt(2009)3%202%20final.pdf.
3 Macfarlane M et al. 2009. A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: skin irritation. Regul Toxicol Pharmacol. Jul;54(2):188-96.
4 Pitman S. (2009, February 20). EU readies for total ban of skin irritation testing on animals. Website accessed on June 13, 2012 at CosmeticsDesign-Europe.com.
5 EC, 2003. Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. Official Journal of the European Union L 66, 26–35. Website accessed on June 7, 2012 at http://ec.europa.eu/consumers/sectors/cosmetics/animal-testing/index_en.htm.
6 Eskes C et al. 2012. Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations. Regul Toxicol Pharmacol. Mar;62(2):393-403.
7 Macfarlane M et al. 2009. A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: skin irritation. Regul Toxicol Pharmacol. Jul;54(2):188-96.
8 Jírová D et al. 2010. Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data. Contact Dermatitis. Feb;62(2):109-16.
9 Cotovio J et al. 2007. In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy Results and performances with 184 cosmetic ingredients. AATEX. Special Issue 14 351:358.