Skin Irritation
Source: http://www.lionden.com/SkinLayers.jpg. Used with permission.
By using the in vitro skin irritation testing services CeeTox offers, clients can practice the 3Rs (reduce, refine, or replace animal testing) and achieve Amendment 7 and REACH directives by reducing or eliminating the need for in vivo testing. These in vitro assays produce reliable outcomes that supplement or replace in vivo data at reasonable cost.
Skin Irritation refers to the production of reversible damage to the skin following the application of a test substance for up to 4 hours. The principle of the in vitro skin model irritation assay is based on the premise that irritant chemicals are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Moreover, if the cytotoxic effect is absent or weak, a quantifiable amount of inflammatory mediators is released by the epidermis and may be used in a tiered approach to increase the sensitivity of the test.
CeeTox uses validated 3D skin models to test for skin irritation. Either liquids or solids can be tested with the models:
- SkinEthic RHE™ (SkinEthic Corporation)
- EpiDerm™ EPI-200 (MatTek Corporation)
The in vitro models have undergone extensive in-house validation at CeeTox and have a high concordance with in vivo findings.
The skin model irritation assay is based on the premise that irritant chemicals are able to penetrate the stratum corneum (see above) by diffusion and are cytotoxic to the cells in the underlying layers. The viability of these cells is determined by the MTT assay, which measures total activity of a cell population e.g., as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), which correlates with the total number and/or vitality of living cells.
Moreover, if the cytotoxic effect measured by the MTT assay is absent or weak, a tiered approach may be used to increase the sensitivity of the test by measuring inflammatory mediators that are released by the epidermis. Testing is done for release of Interleukin 1 alpha, a vertebrate cytokine that is especially important in inducing inflammatory responses. Human keratinocytes express and release large amounts of IL-1α.
In addition, histology can be performed to visualize the morphological changes to the models caused by the test compounds. The histological evaluation improves interpretations and reduces false negatives.
CeeTox has three irritation assays available, as well as a range finding assay. All three irritation methods can be used for prediction of R38 class and also (in line with the 2008 ESAC Statement) they also can reliably distinguish between GHS Class 2 and NI.
EpiSkin™ Skin Irritation Test (SIT) – R38 Reference
- Cell Model: EpiSkin RHE (SkinEthic Laboratories)
- Assays Performed:
- MTT reduction
- IL-1α release (optional)
- H&E stained histology slide (optional)
- Controls: MTT direct reduction, water-killed (if needed), blank, positive, negative; R38 reference
- Exposure: 15 min.
- Incubation: 42 hours
- Number of Replicates: 3
- Standard Turn Around Time: 3 weeks from sample and tissue receipt
- Scientific Endorsement: ECVAM
- International Regulatory Acceptance: Draft OECD Test Guideline
- National/ Regional Regulatory Acceptance: EU test method B.46 in COM regulation 440/2008/EC
EpiDerm™ Standard Skin Irritation Test
Standard skin irritation assay using standard MatTek protocol with 1 time point
- Cell Model: EPI-200 (MatTek Corporation)
- Assays Performed:
- Solubility Assay
- MTT reduction
- IL-1α release (optional)
- H&E stained histology slide (optional)
- Controls: MTT direct reduction, water-killed (if needed), blank, positive, negative
- Exposure: 60 min.
- Incubation: 42 hours
- Number of Replicates: 3
- Standard Turn Around Time: 3 weeks from sample and tissue receipt
- Scientific Endorsement: ECVAM
- International Regulatory Acceptance: Draft OECD Test Guideline
- National/ Regional Regulatory Acceptance: EU test method B.46 in COM regulation 440/2008/EC
SkinEthic RHE™ Skin Irritation Test
Standard skin irritation assay using the standard SkinEthic protocol with 1 time point
- Cell Model: EpiSkin RHE (SkinEthic Laboratories)
- Assays Performed:
- Solubility Assay
- MTT reduction
- IL-1α release (optional)
- H&E stained histology slide (optional)
- Controls: MTT direct reduction, water-killed (if needed), blank, positive, negative
- Exposure: 42 min.
- Incubation: 42 hours
- Number of Replicates: 3
- Standard Turn Around Time: 4 weeks from sample and tissue receipt
- Scientific Endorsement: ECVAM
- International Regulatory Acceptance: Draft OECD Test Guideline
- National/ Regional Regulatory Acceptance: EU test method B.46 in COM regulation 440/2008/EC
EpiDerm™ Skin Irritation Range Finding Assay
This assay uses the standard MatTek protocol but uses 3-5 exposure times to add additional understanding of the potential for skin irritation over a longer exposure time than 1 hour.
- Cell Model: EPI-200 (MatTek Corporation)
- Assays Performed:
- Solubility Assay
- MTT reduction
- IL-1α release (optional)
- H&E stained histology slide (optional)
- Controls: MTT direct reduction, water-killed (if needed), blank, positive, negative
- Exposure: 3-5 exposures from 1 hour to 48 hours
- Incubation: 42 hours
- Number of Replicates: 3
- Standard Turn Around Time: 3 weeks from sample and tissue receipt
- Scientific Endorsement: ECVAM (for standard protocol)
- International Regulatory Acceptance: Uses same protocol as the draft OECD Test Guideline plus adds extra times points
- National/ Regional Regulatory Acceptance: Uses same protocol as EU test method B.46 in COM regulation 440/2008/EC plus adds extra time points
Let CeeTox scientists become an extension of your R&D team when we put our 10+ years of experience to work for you interpreting the data that enables you to make the best decisions and bring safer products to market faster and at lower cost.
Literature Cited:
European Commission Joint Research Centre, Institute for Health and Consumer Protection ,
In-vitro Toxicology Unit, European Centre for the Validation of Alternative Methods (2008) STATEMENT ON THE SCIENTIFIC VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION TESTING(ECVAM) http://ecvam-dbalm.jrc.it/ft_doc/ESAC_Statement_SkinEthic+EpiDerm%20FINAL%200812-01.pdf
Hayden, P. (2007) The Way Forward for In Vitro Skin Irritation Testing. www.alttox.org/ttrc/toxicity-tests/skin-irritation/way-forward/hayden
Harvell, J.D., Lamminstausta, K, Maibach H.I. (1995) Irritant contact dermatitis in: Guin J.D. Practical Contact Dermatitis McGraw-Hill New York, 7-18.
OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Human Skin Model Test http://www.oecd.org/dataoecd/21/56/40793105.doc
For more information visit www.ceetox.com or email info@ceetox.com