As most of the industry is aware, regulation No. 1907/2006 has gone into effect, better known as REACH, or the Registration, Evaluation and Authorization of Chemicals. A complete listing of the directive was published Dec. 30, 2006, in the Official Journal of the European Union, and at over 800 pages, it may require a few cups of coffee to review. In brief, however, REACH is a program designed to improve upon the old method of regulating chemicals in the European Union (EU).*
Under the old EU method, only new chemicals or chemicals introduced after the year 1981 were required to undergo safety testing. Established chemicals or those introduced prior to 1981 were exempt from testing requirements, unless a local European authority deemed testing as necessary. Thus, the old method put the burden of demanding safety information for an established chemical on local authorities. However, with the new REACH regulations, manufacturers are now required to provide safety information on all chemicals they produce that are sold, manufactured, or imported into the EU in a quantity that is greater than or equal to one ton per year. This means that safety data must now be collected for over 30,000 existing and established chemicals.1
The idea of gathering data on so many chemical substances is staggering, to say the least. Even with companies sharing data on common chemicals and using alternative forms of safety assessment such as computer based quantitative structure-activity relationship (QSARs) analysis, it has been estimated that several million animals may need to be sacrificed to screen all of the presently existing chemicals on the market.1
Luckily, the authors of the REACH directive had the foresight to recognize the impact of such a large amount of testing on animal use and mandated that in vitro testing methods should be used as animal testing alternatives whenever possible.2
Despite the mandate for emphasizing nonanimal testing methods, the current list of validated in vitro alternative animal tests is short. However, since the REACH regulations have placed definite time limits on screening the list of existing chemicals, the process of validating in vitro alternatives is accelerating.
As the new REACH regulations go into effect and new testing methods are being introduced, a period of confusion has settled on the chemical industry as to which are acceptable testing methods. Thus, this article begins a series that will provide an overview of some of the currently validated in vitro methods that are alternatives to animal testing, or alternative methods to animal testing that are being considered for validation.
Alternative Methods to Assess Skin Corrosion Potential
The corrosive potential of a chemical is an important concern when determining how chemicals will be packaged, transported and handled. Since the skin is the prime site for exposure to corrosive chemicals, the traditional animal based model to assess the corrosive potential of a chemical has been a Draize rabbit test (see the "Draize rabbit skin test" sidebar at the end of this article). However, in 2004 the Organization for Economic Co-Operation and Development (OECD) set out some guidelines for in vitro epidermal models of corrosive potential testing to replace animal based models.3
The guidelines for the construction of the model require that the epidermal model should be made using human keratinocytes and the keratinocytes should be cultured so that they form a three dimensional, multilayered structure of viable cells similar to the human epidermis. The epidermal model should have a functional stratum corneum with chemical and structural properties that provide the culture with a barrier function that is similar to human skin, and the epidermal model also should be grown in a manner where materials that are applied topically to the stratum corneum can only access the viable lower layers of the tissues by penetrating through the stratum corneum and not flowing around the edges of the epidermal model. In addition, the epidermal model should be prepared in such a way that it is free from contaminating microorganisms.
In addition to the construction guidelines, guidelines regarding how the model should function also were given. These functional guidelines state that the metabolic activity of the tissue should be measured using a vital dye, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and there should be sufficient difference in the optical density of the dye extracted from negative control tissues and the extraction solvent alone. Also, the barrier function of the stratum corneum should distinguish between chemicals of differing corrosive potential and resist the immediate penetration of cytotoxic chemicals. The results obtained from the epidermal model should be consistent and reproducible with respect to the same chemical over time.
Currently there are commercially available skin tissue models that meet the OECD guidelines and have been validated by ECVAM for the in vitro testing of chemical corrosive potential.a,b The basis for this method is the observation that corrosive chemicals are cytotoxic to the viable layers of the epidermis after a short term exposure. In fact, the basic protocol for one of the available tissue modelsa uses just two exposure times. The materials to be screened for corrosive potential are topically applied to the tissues for three minutes and to a second set of tissues for one hour, followed by immediate determination of the cytotoxic effect.
Cytotoxicity is determined through the use of the vital dye MTT. In the tissues, viable keratinocytes will take up MTT, and the reduction of MTT by mitochondrial enzymes results in the formation of insoluble purple formazan crystals. The purple formazan crystals can be extracted with isopropanol and quantified spectrophotometrically. The intensity of the purple color is directly proportional to the number of viable keratinocytes in the tissue, and inversely proportional to the cytotoxicity of the test material. Thus, corrosive materials will reduce the number of viable cells, which in turn will result in less of the purple formazan crystal being formed in the MTT assay. This will be shown quantitatively by a low absorbance measurement of the extracted formazan solution for tissues treated with corrosive materials.4
The absorbance data from the MTT assay can easily be converted into a measurement of tissue viability. Since a negative control of distilled water being applied to tissues is included in the experiment, the average absorbance measurement for the MTT extracted from the negative control tissues can be used to represent 100% tissue viability, meaning all of the keratinocytes are alive and unharmed. Tissue viability can be calculated using the following formula:
Percent Viability = MTT absorbance of treated tissues/mean MTT absorbance of untreated tissues x 100
By dividing the absorbance measurement of the MTT extract from tissues treated with the materials being tested for corrosive potential by the average absorbance value of the MTT extract from negative control tissues, the result will give an index of the viability of the test material treated tissues. If the absorbance measurement of the MTT extract from the tissues treated with the test material is half the absorbance of the negative control extracts, then the tissue viability was reduced to 50%. This means that the test material had a cytotoxic effect and reduced the number of viable keratinocytes in the skin tissue model by half.
Once the viability of the tissues has been determined at each time point, the final classification of a test material as corrosive or noncorrosive with the tissue modela is determined (Table 1).
The in vitro skin models have proven themselves to be a most useful in vitro tool for testing the skin corrosion potential of chemicals. A follow-up column will expand on the function of skin tissue equivalents and discuss their usefulness as alternative in vitro tests for two additional markers: skin irritation and phototoxicity. Stay tuned.
Send correspondence to Robert Holz.
1. T Hofer et al, Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation, Archives of Toxicol 78(10), 549-564 (2004)
2. C Grindon, R Combes, MT Cronin, DW Roberts and J Garrod, A review of the status of alternative approaches to animal testing and the development of integrated testing strategies for assessing the toxicity of chemicals under REACH: A summary of a DEFRA-funded project conducted by Liverpool John Moores University and FRAME, Alternatives to Laboratory Animals 34, Suppl. 1, 149-158 (2006)
3. OECD Guidelines for the Testing of Chemicals,No. 431: In Vitro Skin Corrosion: Human Skin Model Test. Paris, France: OECD, 8 p. (2004a)
4. M Liebsch, D Traue, C Barrabas, H Spielmann,P Uphill, S Wilkins, JP McPherson, C Wiemann, T Kaufmann, M Remmele, HG Holzhütter, The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA 28:371-401 (2000)
5. JH Draize, G Woodward and HO Clavery, Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J of Pharmacology and Experimental Therapeutics 82 377-390 (1944)
6. MK Robinson, JP McFadden and DA Basketter, Validity and ethics of the human 4-h patch test as an alternative to assess acute skin irritation potential, Contact Dermatitis 45(1) 1-12 (2001)
7. L Phillips, M Steinberg, HI Mailbach, WA Akers, A comparison of rabbit and human skin response to certain irritants, Toxicol Appl Pharmacol 21 369-382 (1972)
8. MK Robinson, C Cohen, AB de Fraissinette, M Ponec, E Whittle, JH Fentem, Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products, Food Chem Toxicol 40(5) 573-592 (2002)
Table 1. Mean tissue viability (percent of negative control)
|< 50% at 3 min||corrosive|
|> 50% at 3 min and < 15% at 1 hr||corrosive|
|> 50% at 3 min and > 15% at 1 hr||noncorrosive|
Draize rabbit skin test
The Draize rabbit skin test can be used to determine if a topically applied material is a corrosive (causes irreversible damage to the skin), an irritant (causes reversible damage to the skin) or a non-irritant. The general procedure for the test is to apply the chemical to be tested directly onto the shaved skin of an albino rabbit and then grade the skin response at various time intervals.5 If the chemical produces irreversible skin lesions that penetrate into the dermis with less than four hours of exposure then the chemical can be classified as corrosive.6 While the Draize rabbit skin test has been in use for over 50 years, it has long been criticized on its ability to accurately predict if a chemical will be irritating to humans.7 In addition, animal welfare groups often dub it as an inhumane testing method.8
*This article appears in the December edition of Cosmetics & Toiletries magazine
aEpiDerm is a product of MatTek Corp.
bSkinethic Human Skin Model is a product of Skinethic