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In this article we will discuss about:- 1. Introduction to Skin Toxicity 2. Types of Toxic Effects 3. Effects on Epidermal Adnexa 4. Skin Testing Methods 5. Evaluation of Skin Tests.
Introduction to Skin Toxicity:
The body of all animals, including humans, is almost entirely covered by a layer termed skin which is usually exposed to a variety of physical and chemical exposures such as cosmetics, household products, topical medication, industrial as well as environmental pollutants, radiations etc. Such an exposure of the epidermis and the dermis of the skin rests over the subcutaneous tissue.
The epidermis of humans is relatively thin, averaging 0.1-0.2 mm in thickness, whereas the dermis is about 2 mm thick. These two layers are separated by a basement membrane. The epidermis in turn consists of a basal cell layer (stratum germinativum), which provides new cells to other layers. These new cells become prickle cells (stratum spinosum) and, later, the granular cells (stratum granulosum).
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The nuclei in these cells disintegrate and dissolve. In addition, these cells produce keratohydrin, which later becomes keratin in the outermost stratum corneum. This layer is gradually shed. The epidermis also contains melanocytes, which produce pigments, the Langerhans cells, which act as macrophages, and lymphocytes. The latter two types of cells are involved in immune responses. The epidermis thus forms protective covering of the body.
The dermis is primarily composed of collagen and elastin which are important structures to support the skin. In this layer, there are different types of cells, the most abundant being the fibroblasts, which participate in the biosynthesis of fibrous proteins and ground substances such as hyaluronic acid, chondroitin sulphates, and mucopolysaccharides. The other types of cells include fat cells, macrophages, histiocytes, and mast cells.
There are, in addition, various other structures. These include the hair follicles, sweat glands, sebaceous glands, small blood vessels, and neural elements, including Meissner’s corpuscles.
Types of Toxic Effects:
Different types of effects may result from dermal exposure to toxicants. Most of the effects involve the skin itself, but some of them affect its appendages — hair, sebaceous glands, and sweat glands.
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Actually, irritation is a local reaction of the skin to such chemicals as strong alkalis, acids, solvents, soaps, detergents and environmental contaminants. It ranges in severity from hyperemia, edema, and vesiculation to ulceration (corrosion). Primary irritation occurs at the site of contact and, in general, on the first contact. It is thus different from sensitization.
The skin may show little or no reaction on contact with chemical. However, a reaction occurs after a subsequent exposure to the chemical. This delayed reaction, with a latent period ranging from a few days to years, apparently involve a complex immune mechanism. After penetration through the epidermis, the toxicant forms a covalent bond with certain proteins.
A sensitization reaction often results from topical medications such as antibiotics, antiseptics, local anesthetics, antihistamines and stabilizers (e.g., ethylenediamine). Other substances include plants (e.g., poison ivy), metal compounds, dyes, cosmetics and industrial pollutants.
iii. Phototoxicity and Photoallergy:
These skin reactions are almost similar in that both are light-induced and may follow either systemic administration or topical application of the offending chemical. However, phototoxicity involves no immune reactions, whereas photoallergy does.
Phototoxicity is much more common than photoallergy. The very commonly phototoxic chemicals in humans are chlorothiazides, phenothiazines, coal tar, pyridine, acridine, sulfonamides and phenanthrene. The skin reaction consists of a delayed erythema, followed by hyper- pigmentation and desquamation.
The well-known photoallergic chemicals are multifungin, promethazine, cyclamates, thiazides, chlordiazepoxide etc. Many compounds are, therefore, photoallergy and phototoxic.
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A special type of reaction, involving mainly the skin in contact with the offending physical or chemical agents, but sometimes there are also systemic effects. The systemic effects include generalized urticarial and anaphylactoid reactions.
It has been known for over two centuries that soots may cause skin cancer. Recent studies confirm that soots and related substances, e.g., creosote oil, coal tars, shale oils, etc., cause cancers of the skin and other sites in animals and humans (International Agency for Research on Cancer, 1979).
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Various polycylic aromatic hydrocarbons (e.g., benzo (a) pyrene) and heterocyclic compounds (e.g., benz (c) acridine) are known to induce skin cancer after topical application on animals (IARC, 1973).
Effects on Epidermal Adnexa:
Various antimitotic agents used in cancer chemotherapy may induce hair loss. These agents hit at the anagen phase of hair growth. The affected hair sheds after about two weeks of therapy, but hair growth takes about two months after the withdrawal of therapy.
Various other medications are known to cause loss of hair by altering hair follicles in anagen phase to telogen phase. In such cases, hair shedding normally starts 2-4 months after therapy. The medications involved in this type of hair loss include oral contraceptives, anticoagulants, propanol and tripanol.
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These glands secrete fat through the expulsion of their lipid laden cells and are, therefore, known as holocrine. Their activity is hormone-dependent.
Various chlorinated aromatic hydrocarbons can cause various skin lesions, including chloracne. The severity of acne varies, but it has been confined chiefly to occupational workers.
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Sweating serves useful physiologic functions such as regulation of body temperature. Blockage of the sweat ducts, a disorder known as miliaria, may occur after topical application of 95% chloroform and phenol. Systematically, atabrine may cause anhidrosis. A number of antimitotic agents can cause necrosis of the sweat glands.
Skin Testing Methods:
Selection of Animals:
To study the effect of toxicants on the skin, albino rabbits are usually preferred although albino guinea- pigs, white mice and others may also be used.
1. Test for Primary Irritation:
To measure the primary irritation of the skin a patch-test technique is adopted on the hair-free abraded and intact skin of the albino rabbit. This method was outlined by Draize, 1959. The method consists of introducing 0.5 ml or 0.5 gm of the test substance, under a one square-inch (6.25 sq. cm.) patch.
The animals are immobilized in an animal holder with patches secured in place by adhesive tape. The entire trunk of the animal is then wrapped with rubberized cloth for the entire 24 hour period of exposure. This latter procedure aids in maintaining the test patches in position and, in addition, retards the evaporation of volatile chemicals.
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After 24 hrs. of exposure the patches are removed and the resulting reactions are evaluated on the basis of scores as cited in Table 16.4:
Readings are also made after 72hrs and the final score represents an average of 24 and 72hr readings. An equal number of exposures are made on areas of skin which have been abraded earlier. The abrasions are minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma (i.e., not sufficiently deep to produce bleeding).
The total erythema and edema scores are added in both the 24 and 72hr readings and the mean of the scores for intact and abraded skin are combined. This combined average is referred to as the primary irritation index.
Chemical compounds producing combined averages (primary irritation index) of 2 or less are only mildly irritants, whereas with indices from 2 to 5 are moderate irritants, and those with scores above 6 are considered severe irritants.
2. Test for Sensitization Reaction:
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The procedure for sensitization reaction was described by Draize in 1959. In this procedure guinea-pigs are given 10 repeated intradermal injections of the chemical on the flank and a challenging dose on the other flank after a 10 to 14 day resting period. A greater reaction after the challenging dose, in comparison with that after the sensitizing doses, indicates sensitization.
However, Draize test is considered insufficiently sensitive to identify allergic potential. So much so, the intradermal injection is regarded inappropriate to assess the safety of chemical to be applied on the surface of the skin.
Various modifications have thus been proposed:
Freund Complete Adjuvant Test:
This test involves the use of Freund adjuvant in order to enhance the sensitivity. Furthermore, in challenging the treated animals, four concentrations of the chemical are given, thus providing a basis for quantitative estimation. However, as in Draize test, the test material is given by intradermal injection.
One of these methods (Buehler test) stipulates that the test material, after dilution, be applied topically to the skin and held in place for 6 hours by means of an occlusive patch for the induction and challenge. In the other, the open epicutaneous test, the undiluted compound and/or its progressive dilutions are applied to the skin, which remains uncovered.
3. Test for Phototoxicity and Photoallergy:
For the dectection of phototoxicity tests, hairless mouse, rabbit and guinea-pigs are used as test animals. The substance to be tested may be administered topically or by a systemic route. The reaction of the skin to non-erythrogenic light (wavelength greater than 320 nm) is then determined. Significant erythema, compared to controls, indicates phototoxicity.
For the examination of photoallergy, albino guinea pigs are particularly useful. The procedure involves, in principle, an induction of photo- sensitization by repeatedly applying a small amount of the chemical on a shaved and depilated area of the skin and exposing that area to appropriate ultraviolet rays. After 3 week interval, the guinea-pigs are exposed to the chemical and the UV rays to elicit photoallergy.
4. Test for Contact Urticaria:
Hunziker (1977) performed the patch test on the flank and nipples of guinea-pigs and described the gross and histologic changes produced by various chemicals viz., dinitrochlorobenzene. The open patch test can be applied to human patients suspected of being susceptible to the chemical.
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Any immunologic involvement can be shown by the passive transfer test, in which 0.1 ml fresh serum from the patient is injected intradermally into the forearm of a volunteer and challenged 24 hours later by applying the suspect chemical to the injection site.
For the detection of cutaneous cancer, the mouse is the commonly used animal.
The procedure involves topical application of the chemical on a shaved area of the skin. The chemical, if a liquid, is applied directly. Otherwise it is dissolved or suspended in a suitable vehicle. The skin painting is usually done once a week or more frequently. It is advisable to include a vehicle control group as well as a positive control group, which is treated with a known skin carcinogen such as benzo (a) pyrene.
This is usually performed in guinea-pigs. The chemical is administered by the same route as that to be encountered in human exposure conditions. A vehicle control group is included to assist in the interpretation of the results.
In this test, rabbit is usually preferred as the test animal. In 1968, Kligman and Katz described a test according to which the test substance is inserted in the external ear canal of rabbits (the test animal). Positive reactions are marked by thickened mounds on the follicles, caused by distention with horny materials.
Evaluation of Skin Tests:
The aforementioned testing procedures have been widely used, despite certain drawbacks.
Seeking to improve on them, Middleton (1981) has proposed the following new approaches, which are designed to measure more specific biological and biochemical events:
i. Alterations in the normal differentiation pattern of epidermis, which are demonstrable by measuring in different layers, the contents of phospholipids, keratin, and a histidine-rich protein.
ii. Alterations in epidermal cell proliferation as shown by the mitotic index among basal cells and the rate of DNA synthesis.
iii. Alterations in cellular viability as determined by enzyme leakage.
Presently these approaches still require considerable further investigations to ascertain their predictive value and to work out the optimum procedures.
