The following guidelines present an in vitro alternative, which uses excised animal or human skin, to experiments conducted in vivo on animals or humans for the assessment of dermal absorption and percutaneous penetration. Both aspects are essential for the risk assessment of chemicals which may contact and subsequently penetrate the skin.There are scientific and ethical reasons for this type of test. Animal skin is obtainable in sufficient quantities to allow replication of experiments. Extrapolation of data obtained from appropriate animal skin is an acceptable alternative but excised human skin obtained from surgery may be used when available. Furthermore, no risk or harm for living creatures is associated with this type of test.These guidelines were developed by a Task Force of COLIPA, the European Cosmetic, Toiletry and Perfumery Association (COLIPA, 1995). Each of the members of the Task Force has several years' experience in the assessment of dermal absorption and percutaneous penetration of chemicals used in products.These guidelines utilize the principles outlined in the ‘OECD New Guideline Proposal on In Vitro Percutaneous Absorption of Chemicals’.
Scalp penetration of diaminoanisole (DAA), p-phenylenediamine (PPD), and N4,N4-Bis-(2-hydroxyethyl)-N'-methyl-2-nitro-p-phenylenediamine (HC Blue #1) that occurs under conditions of hair dye usage was evaluated for both Rhesus monkey and man using 14C labeled materials. Both specieis showed a remarkably similar pattern of dye penetration. Thus the average dose excretion of DAA in the Rhesus monkey was 0.02% and in man 0.015%; PPD excretion in monkey and man was 0.14%; and HC Blue #1 excretion in the monkey was 0.12% and in man, 0.09%.
The human skin grafted athymic nude mouse, pig skin grafted athymic nude mouse, hairless dog, and weanling Yorkshire pig were evaluated as models for predicting skin penetration in man. Nine radiolabeled compounds previously tested on man were applied topically (4 micrograms/cm2) to each animal. These compounds included caffeine, benzoic acid, N,N-diethyl-m-toluamide, three steroids, and three insecticides. To correct for incomplete excretion of the label following topical absorption, percentage penetration was calculated by dividing the percentage of the topically applied radioactive dose recovered in the excreta by the corresponding percentage after parenteral administration and multiplication by 100. In the case of the grafted athymic nude mouse, calculated values of percentage penetration were confirmed because significant correlations (r = 0.78 for the human skin grafted athymic nude mouse and r = 0.97 for the pig skin grafted athymic nude mouse) were found between the calculated values and percentage penetration determined by summing radioactivity recovered in the urine, feces, tissues, and carcass. The results revealed a significant correlation between human skin grafted athymic nude mouse values and human values (r = 0.74, p = 0.05), and between weanling Yorkshire pig values and human values (r = 0.83, p = 0.05). In contrast, no significant correlation existed between human values and those of the hairless dog and pig skin grafted athymic nude mouse. The disposition of radioactivity following topical application of the radiolabeled nerve agent analog ( diisopropylfluorophosphonate ) and simulant (diethyl malonate) was determined in the weanling pig and the human skin grafted athymic nude mouse.(ABSTRACT TRUNCATED AT 250 WORDS)
The percutaneous absorption of compounds through the skin of selected animal species was compared to that occurring with human skin, using in vitro diffusion cell techniques. The permeability of back skin of the following animals was examined: Hormel miniature pigs, Osborne-Mendel rats, NIH hairless mice, and Swiss mice. Benzoic acid, acetylsalicylic acid, and urea, dissolved in a petrolatum vehicle, were applied and permeability constants were determined. With the faster penetrating compounds, benzoic acid and acetylsalicylic acid, mouse skin and hairless mouse skin were similar and were the most permeable. Little difference was observed in acetylsalicylic acid absorption of human, pig, and rat skin. Pig skin and hairless mouse skin were the best animal models for the slower penetrating urea. The thicknesses of the stratum corneum, epidermis, and whole skin were determined from microtome sections prepared from frozen skin. In addition, the density of hair follicles in each type of skin was determined. In general terms, thickness of the stratum corneum was found to be important: the thickest stratum corneum was that of pig skin and the thinnest was that of the mouse. Often however, differences in permeability and stratum corneum thickness did not correlate on a 1:1 basis, which can be indicative of differences in structure of the different types of skin. The animal model of choice is dependent on the compound. For benzoic acid and acetylsalicylic acid, the pig and rat are good models for human skin. In the case of slow absorbing compounds, such as urea, diffusion through appendages in the skin may make undesirable the use of skin of densely haired animals.
This paper presents an in vitro technique to analyse percutaneous penetration and dermal absorption of hair dyes, topically applied to excised pig skin. Representative examples are given by the radio-labelled hair dyes p-phenylenediamine and bis-(5-amino-1-hydroxyphenyl)-methane. Both compounds were assessed under simulated use conditions and were analysed in representative formulations including the specific conditions for oxidation hair dyes. To be able to differentiate between topically adsorbed and systemically available amounts, the bioavailability of the hair dyes is defined as the amount penetrated and/or remaining in the exposed skin after removing the stratum corneum. Less than 1% of the assessed topically applied dyes was found to be bioavailable in the presence of hydrogen peroxide, typically added to oxidation hair dyes prior to applications. Compared with published results and unpublished in-house in vivo data, the level of confidence was high. Owing to in-house experience over about 5 years in using excised pig skin for measurements of percutaneous penetration and dermal absorption of hair dyes, the technique was found to be successful and appropriate to reduce the number of test animals normally used for toxicological assessments. The essentials of this technique are actually recommended by the SCCNFP (The Scientific Committee on Cosmetic Products and Non Food Products intended for Consumers) for the safety evaluation of cosmetic ingredients, particularly for hair dyes. The corresponding OECD guideline as well as the guidance document has been drafted and is currently in discussion on the level of the national coordinators.