Overview of animal test methods for skin irritation

Fd Chem. To.~i~. Vol. 23, No. 2. pp. 165-168. 1985 Printed in Great Britain. All rights reserved

0278-6915/85 $3.00 + 0.00 Copyright © 1985 Pergamon Press Ltd

OVERVIEW OF ANIMAL TEST METHODS FOR SKIN IRRITATION W. GFELLER,W. KOBEL AND G. SEIFERT Ciba-Geigy Ltd, 4002 Basel, Switzerland Abstract--Determining the irritant effects of chemicals in experimental animals provides the public with information on and protection against possible hazards that may arise from exposure of the human skin. Furthermore. the data obtained are used for registration and classification purposes. This paper reviews the methods proposed in the most important guidelines (OECD, EEC and FIFRA guidelines) and points out the critical experimental points. It shows that the introduction of OECD Guideline No. 404 can lead to a reduction in the number of test animals used for skin irritation testing.

Introduction Testing for potential hazard to the skin from acute exposure is routinely required for several classes of chemicals to which humans may be exposed accidentally or intentionally. These chemicals include cosmetics, non-cosmetic skin preparations, pesticides, industr;al chemicals, dyestuffs, detergents and household substances. Several regulatory authorities have issued guidelines on the conduct of animal tests for skin irritation and corrosion. Information from such tests is required for registration, classification and labelling purposes. The complexity of the field is illustrated by reference to the USA. where guidelines have been issued in connection with the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA; EPA, 1978 & 1982), the Toxic Substances Control Act (TSCA; EPA, 1979) and the Federal Hazardous Substances Act (FHSA; CPSC, 1981), as well as by the National Academy of Sciences-National Research Council (NAS-NRC, 1977) and the Interagency Regulatory Liaison Group (IRLG, 1981). In Europe, besides the OECD (1981) and EEC (1979) guidelines, several countries have issued national guidelines. The aim of this paper is to give a short overview on the relevant testing methods, to present the methods for assessing and calculating the findings, to discuss the relevance of the test methods and to consider the consequences of the new international guidelines on the numbers of animals used. A reliable test for skin irritation should provide a means for differentiating among substances that will cause different degrees of irritation or corrosion of the skin. In this context, qrritation' is the local inflammatory response of normal living skin to direct injury by single, repeated or prolonged contact with a chemical agent without the involvement of an immunological

mechanism. The macroscopic manifestations are erythema and oedema. 'Corrosion' is a direct chemical action on normal living skin resulting in disintegration and irreversible alteration of the skin at the site of contact Its important manifestations are ulceration, necrosis and scar formation. All the currently established procedures for skin irritation testing are based on the method proposed by Draize, Woodard & Calvery (1944). However, the guidelines proposed under FIFRA (EPA, 1978) stated that substances with a pH of 1-3 or 12-14 need not be tested on animals. Such substances are considered to be corrosive. Slightly more stringent limits were given in the new edition of the FIFRA guidelines (EPA, 1982). as well as in the OECD (1981) guidelines, which exempt compounds with a pH below 2.0 or above 11.5 from testing. In addition highly toxic materials need not be tested. These exemptions seem to be very reasonable, but Guillot, Gaillard~ Gonnet & Clement (1980) demonstrated that these limits are not absolute and in particular cases animal testing may be indicated.

Experimental aspects

To clarify the differences between the guidelines, the points of experimental significance are summarized in Table 1. The preferred test animal is the rabbit and usually an albino strain is used. Besides this species, mice and guinea-pigs have also been used. Roudabush, Terhaar, Fassett & Dziuba (1965) reported ttiat they found relatively little interspecies difference between the rabbit and the guinea-pig but both models had a tendency to exaggerate the response. A similar conclusion can be drawn from the work of Davies, Harper & Kynoch (1972), who compared several more animal species in this respect. The test substance can be applied to intact or Abbreviations: CPSC = Consumer Product Safety abraded skin (prepared by scarification of the Commission; EEC = European Economic Community; stratum corneum without injury to the dermis). The EPA =EnvironmentaI Protection Agency: FHSA = stratum corneum is an effective barrier which is Federal Hazardous Substances Act; FIFRA--removed by scarification. The resulting condition Federal Insecticide. Fungicide and Rodenticide Act; simulates damaged skin. Generally a more severe IRLG = Interagency Regulatory Liaison Group; reaction can be expected from the abraded skin NAS = National Academy of Sciences; NRC= National Research Council: OECD = Organisation for (Nixon, Tyson & Wertz, 1975), making the test more sensitive. This may be a desirable effect since some Economic Co-operation and Development; PII= formulations, such as cosmetics and other skin Primary irritation index.

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W. GFELLERet al. Table 1. Comparison of test methods Test Variable

Draize

OECD/EEC

Occlusive dressing Access to patch Exposure time (hr) Washing Abrasion

Albino rabbit NS NS At least 3 0.5 ml undiluted 0.5g Gauze Non-irritant, held loosely by semi-occlusivedressing May be used To be prevented 4 Allowed Not required

Examination times (hrt) Scoring Corrosion

0.5-1, 24, 48 and 72 Draize score To be noted

Species Age Weight No./test group Dose: liquids solids or semisolids Patch material Patch tape

As OECD NS NS 6 As OECD 0"5 g* Gauze: 2 layers thick Adhesive. entire trunk wrapped with impervious(rubberized) cloth NS NS 24 NS Abrasion and normal skin for each animal 0 and 48 As OECD Not considered

FIFRA As OECD NS NS 6 As OECD 0.5g As OECD As OECD As OECD As OECD 4 As OECD As OECD As OECD As OECD As OECD

NS --- Not specified *Solids to be dissolved in appropriate solvent. "rAfterpatch removal. preparations but not technical materials, are intended to be applied directly to intact or injured skin. The investigations of Vinegar (1979) demonstrated a regional variation in primary skin irritation in rabbits. The irritation indices from applications in the abdominal region were significantly higher than those in the dorsal region. This may be due to the difference in the thickness of skin. Most guidelines specify the dorsal region as the application site; since t h e animal has practically no direct access to this part of the body, ingestion or inhalation of the test material is thus prevented. The location of the application site is important with regard to comparing results of different chemicals. The test material is generally applied in a dose of 0.5 g or 0.5 ml spread on a surface area of 6 cm 2. Liquids are applied as such and present no problems. Semisolids and solids need to be moistened or dissolved to ensure g o o d contact with the skin. The techniques used vary widely. A solid test c o m p o u n d can be applied dry onto premoistened skin, can be applied dry onto dry skin and be covered with a moistened gauze or filter patch or can be applied as a slurry. It is not clear how much these technical differences influence the test result; reports on studies of this question are in parts contradictory (McCreesh & Steinberg, 1977; Sullivan, Strausburg & Kapp, 1975). N o r m a l l y the a m o u n t of water or vehicle used is equal (0.5 ml) to the quantity of material to be tested. Most of the guidelines do not specify exactly the technique to be applied. C o m m o n l y recommended for covering the test site is the gauze patch or other semi-absorbent inert material. The technique used to fix any patch is of primary importance. Recommendations differ as to whether the patch should be kept under an occlusive or a semi-occlusive cover. Occlusive covering of the test patch enhances the skin response by hydration of the stratum corneum, by an increase of the skin temperature and by an increase of the skin surface area by 37 ~'/~,. Additionally, the test material cannot evaporate. This results in a higher skin sensitivity

and consequently in a more severe irritation. This may be advantageous in the detection of mild irritants, but should be taken into account in the testing of compounds with, for example, a high content of surfactants or alcohols. For such compounds the open type of application may be advantageous, although practical difficulties exist. So far, no guideline has recommended the use of any of the chamber techniques developed by several teams, such as the KI chamber (Kurokawa. Iwamota & Hasegawa, 1980) and the Duhring chamber (Frosch & Kligman, 1979). The older guidelines required an exposure period of 24 hours. Since this requirement did not reflect a realistic situation in most cases, practically all test procedures now specify an exposure period of 4 hours. When comparing the 24-hour with the 4-hour exposure, Gilman, Evans & De Salva (1978) found a significant difference and considered the longer period of exposure an exaggeration. A group of French workers (Guillot et al. 1980) investigated the same question and found that the 24-hour exposure produced a more severely irritant response in most cases, but the differences were rather small. Evaluation of the findings is based on the inflammatory reactions erythema (and eschar formation) and the development of oedema. Erythema is assessed visually and oedema by very light palpation. The Draize e t al. (1944) scale is the most widely used basis for scoring the response (see Appendix, p. 337 in this issue). Readings have to be taken 0.5-1, 24, 48 and 72 hours after removal of the patch, If the signs of irritation are still detectable at 72 hours further observations are recommended, to assess the reversibility up to a m a x i m u m observation period of 14 days. Assessment and classification It is clear that, in a test with so many variables, it is difficult to give a universal method for the assessment. The authorities issuing test guidelines

Animal tests for skin irritation must have been aware of this problem, since there are few instructions on how to classify the responses. The classic method is to add the erythema and oedema scores and take the average of the 24- and 72-hour readings. This numerical value is called the "primary irritation index" (PII). The Draize proposals (Draize, 1959; Draize et al. 1944) gave rise to the following ranking system correlating response category with PII: negligible response, PII 0.0--0,4; slight, PII 0.5-l.9: moderate, PI1 2.0--4.9; strong, PII 5.0-8.0. There have been numerous modifications of these response categories. The FHSA guidelines distinguish only between non-irritants and irritants with a cut-off point at a score of 5.0. Others have divided the 0-8 score values into as many as six classes. For the FIFRA classification, the findings of the 72hour reading are the most important ones. The EEC recently issued a directive distinguishing between non-irritant, irritant and corrosive compounds (EEC, 1979). A material is considered to be irritant if the mean score of erythema or oedema, read after 24, 48 and 72 hours, is 2.0 or higher or if the respective mean per animal is 2.0 or higher in two of the three animals. Any finding of corrosion leads to the classification "corrosive'. Many attempts have been made to compare results from acute and repeated tests on animals with results from tests performed in man (Brown, 1971; Davies et al. 1972; Gritfith & Buehler, 1977; Marzulli & Maibach, 1975; NAS-NRC, 1977). Analysis of these publications has revealed that the rabbit reacts more sensitively than man to various chemicals and that tests on guinea-pigs may possibly give better agreement with findings in man. However, the guinea-pig has the disadvantages of a much smaller test area and a higher sensitivity to lipids.

Skin testing and animal protection A reduction in the use of animals has followed the introduction of new guidelines for skin testing. Since January 1982 the skin irritation tests have been performed in our laboratory in accordance with the OECD Test Guideline No. 404 (OECD, 1981). This procedure exempts from testing compounds with a pH of 2 or less or 11.5 or more on the assumption that they will be corrosive to the skin. Moreover compounds without adequate physico-chemical characterization are no longer deemed acceptable for irritation testing. In 5 years, animal utilization for skin irritation testing has shown a marked decline in our laboratories; taking the 1979 figure of 1926 animals as 100~, the figures for subsequent years have been 2028 (105.3°~o) in 1980, 1614 (83.8%) in 1981, 1005 (52.5°,0)in 1982 and 729 (37.9,°/o) in 1983. It should be understood that this reduction was due to the different approach to testing and not to a decrease in the compounds tested or in safety requirements. The Plls found in skin irritation tests routinely performed in our laboratories on agrochemicals, dyestuffs and industrial chemicals in the years 1979-81 were collected and the distribution of the scores was examined. The numbers of compounds (expressed in brackets as a percentage of the total) in

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each PII range were as follows: Pll 0.0, 8 (3.1 '~i): PII 0.1-1.0, 65 (25%); 1.1-2.0, 73 (28.1 ~o), 2.1--4.0, 72 (27.7%); 4.1--6.0, 25 (9.6~D; 6.1-8.0. 17 (6.5",). Animals with signs of moderate, slight or minimal local irritation without systemic intoxication do not show signs of pain. Animals with signs of marked and extreme local irritation may show signs of pain, reflected by a curved body position. However, even in cases showing signs of extreme local irritation, food consumption and body-weight development were usually unaffected. It may be concluded that less than 16 °J0 of the test substances submitted for testing over a 2-year period caused clearly recognizable discomfort in the animals used. A similar evaluation has been made by Guillot & Gonnet (1982) for cosmetics. In general only 10 o•~, of the formulations induced reactions. The application of the OECD (1981) guideline will decrease this proportion further.

Final considerations In order to protect humans from substances with irritative potential, skin irritation tests must be performed. The relevant guidelines have reached a relatively high degree of harmonization and permit the use of minimum numbers of animals for skin irritation testing. The chemical industry is aware of the deficiencies of the actual testing procedures and is willing to introduce alternative methods. However new methods have to be acceptable for registration purposes and sensitive enough to characterize a potential irritant. They must also be suitable for testing substances irrespective of their physicochemical properties.

REFERENCES Brown V. K. H. (t971). A comparison of predictive irritation tests with surfactants on human and animal skin. J. Soc. cosmet. Chem. 22, 411. CPSC (1981). CFR Title 16, Subchapter llC--Federal Hazardous Substances Act Regulations, Secs 1500.40 & 1500.41. Davies R. E., Harper K. H. & Hynoch S. R. (i972). Interspecies variation in dermal reactivity. J. Soc. cosmet. Chem. 23, 371. Draize J. H. (1959). Dermal toxicity. In Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. p. 46. Association of Food and Drug Officials of the United States, Austin, TX. Draize J. H., Woodard G. & Calvery H. O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J, Pharmac. exp. Ther. 82, 377. EEC (1979). Council Directive 79/831/EEC (18 September). Annex V, Part B: Methods for the determination of toxicity. Annex VII: 4.1.5 Acute toxicity, skin irritation. Off. J. Europ. Commun. 22, (L259), 10. EPA (1978). Proposed guidelines for registering pesticides in the U.S.; hazard evaluation: humans and domestic animals. Fed. Reg. 43, 37336. EPA (1979). Proposed health effects test standards for Toxic Substances Control Act test rules and proposed Good Laboratory Practice standards for health effects. Fed. Reg, 44, 44054. EPA (1982). Pesticides registration; proposed data requirements. Sec. 158. 135: Toxicology data requirements. Fed. Reg, 47, 53192.

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Frosch P. J. & Kligman A. M. (1979). The Duhring chamber, an improved technique for epicutaneous testing of irritant and allergic reactions. Contact Dermatitis 5, 73. Gilman M. R., Evans R. H. & De Salva S. J. (1978). The influence of concentration, exposure duration and patch occlusivity upon rabbit primary dermal irritation indices. Drug Chem. Toxicol. 1,391. Griffith J. F. & Buehler E. V. (1977). Prediction of skin irritancy and sensitizing potential by testing with animals and man. Proceedings of 3rd Conference on Cutaneous Toxicity, p. 155. Guillot J. P., Gaillard L., Gonnet J. F. & Clement C. (1980). Produits chimiques, tol6rance locale oculaire et cutan6e. IFREB paper to Salon Interchimie 1980. Guillot J. P. & Gonnet J. F. (1982). Lokale Toleranzversuche beim Kaninchen und Meerschweinchen--angewandt auf kosmetische und k6rperhygienische Produkte. SeifenOle-Fette-Wachse 1 ~ (7), 188. IRLG (1981). Recommended Guideline for Acute Dermal Toxicity Test: Testing Standards and Guidelines Work Group. IRLG, Washington, DC. Kurokawa M., Iwamota H. & Hasegawa I. (1980). KIchamber patch test unit. J. Soc. cosmet. Chem. 31, 97. Marzulli F. N. & Maibach H. I. (1975). The rabbit as a model for evaluating skin irritants: a comparison of results obtained on animals and man using repeated skin

exposures. Fd Cosmet. Toxicol. 13, 533. McCreesh A. H. & Steinberg M. (1977). Skin irritation testing in animals. In Adt'ances in Modern Toxicolo~jy. Volume 4. Dermatotoxicology and Pharmacology. Edited by F. N. Marzulli & H. I. Maibach. p. 193. John Wiley. New York. NAS-NRC (1977). Dermal and eye toxicity tests. In Principles and Procedures for Ecaluatin# the Toxicity of Household Substances. NAS Publn no. 1138, p. 23, NAS. Washington, DC. Nixon G. A., Tyson C. A. & Wertz W. C. (1975). Interspecies comparisons of skin irritancy. To.~:ic. appl. Pharmac. 31, 481. OECD (1981). Acute dermal irritation/corrosion. In OECD Guidelines for Testing of Chemicals. Section 4, no. 404. OECD, Paris. Roudabush R. L., Terhaar C. J.. Fassett D. W. & Dziuba S. P. (1965). Comparative acute effects of some chemicals on the skin of rabbits and guinea pigs. Toxic. appl. Pharmac. 7, 559. Sullivan J. B., Strausburg J. C. & Kapp R. W., Jr (1975). A comparative study of dermal reactions using the intact rabbit skin (Abstract). Toxic. appl. Pharmac. 33, 165. Vinegar M. B. (1979). Regional variation in primary skin irritation and corrosivity potentials in rabbits. Toxic. appl. Pharmac. 49, 63.