Human Skin Sensitization Research Articles

3: Final Report on the Safety Assessment of Methylene Chloride

MeCl2 is a volatile compound that, if applied as an aerosol, would not remain on the skin for an extended period of time. However, certain cosmetic products that contain MeCl2 are designed to remain on the skin and thus may expose the skin to MeCl2 for longer time periods. It has been estimated that the use of hair sprays containing MeCl2 could expose the user to MeCl2 concentrations ranging from 1 to 154 μg/liter of air. MeCl2 was irritating to the skin of animals; an antiperspirant and a deodorant containing MeCl2 were mildly irritating to the skin of humans. Limited data obtained from animal and human studies indicated that MeCl2 did not induce sensitization. No data were available on whether MeCl2, when applied in a medium designed to remain on the skin is or is not a sensitizer. MeCl2 does not absorb UV-A or UV-B light; therefore, it is unlikely to be a phototoxic agent. In seven different laboratory studies with sufficient animals to permit meaningful statistical analysis of the data, only the results from a chronic mouse study indicated that MeCl2 is a carcinogen. The results of pharmacokinetic and metabolism studies established that the metabolism of MeCl2 in the mouse differs quantitatively from that in the other species studied, and this difference in metabolism could account for the positive tumorigenic response in the mouse. The available epidemiologic studies did not indicate a difference in cancer rates between exposed and unexposed groups. To conclude that the one positive carcinogenicity study is of greater significance in safety evaluation than the six negative carcinogenicity studies, it would be necessary to exclude the results of the pharmacokinetic, metabolism, and epidemiologic studies that support the conclusion that MeCl2 is not a carcinogen for humans and other animal species. Considering all the data, in particular the pharmacokinetic and metabolism data, it is concluded that the data are insufficient to establish MeCl2 as a human carcinogen. Further, it is also concluded that the positive carcinogenic response obtained in the mouse study cannot be extrapolated to humans. The evidence is that MeCl2 is a carcinogen for mice but is not carcinogenic for rats, hamsters, or humans. Because of the lack of human skin irritation and sensitization data on cosmetic products containing MeCl2, the safety conclusion is restricted to aerosol products in which the duration of exposure (skin contact) is limited by the rapid evaporation of MeCl2 from the skin. On the basis of the data included in this report, it is concluded that MeCl2 is safe for use in cosmetic products designed for brief discontinuous use.

5 Final Report on the Safety Assessment of Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl Alcohol

Cetearyl, Cetyl, Isostearyl, Myristyl, and Behenyl Alcohols are long-chain aliphatic alcohols that are, at most, only slightly toxic when administered orally at doses of 5 g/kg and greater. In acute dermal toxicity studies (rabbits), doses of up to 2.6 g/kg of Cetyl Alcohol and 2.0 g/kg of a product containing 0.8% Myristyl Alcohol were both practically nontoxic. Mild irritation was observed when a cream containing 3.0% Cetearyl Alcohol was applied to the skin of New Zealand albino rabbits. Cetyl Alcohol (50.0% in petrolatum) applied to abraded and intact skin of albino rabbits produced minimal to slight skin irritation. Cetyl Alcohol was considered to be practically nonirritating when instilled into the eyes of albino rabbits. An aerosol antiperspirant containing 3.0% Myristyl Alcohol induced mild to moderate irritation; a moisturizing lotion containing 0.8% Myristyl Alcohol was nonirritating to rabbit eyes. Corneal irritation was reported following an ocular test using a 5.0% Isostearyl Alcohol antiperspirant. Conjunctival irritation was observed 2 and 6 h after instillation of 1.0% Behenyl Alcohol. Isostearyl Alcohol (5.0% in propylene glycol) and an antiperspirant containing 5.0% Isostearyl Alcohol were not sensitizers in guinea pigs. Cetyl Alcohol was not mutagenic in Salmonella typhimurium LT2 mutant strains in the spot test. Clinical skin irritation and sensitization studies of product formulations containing up to 8.4% Cetyl Alcohol produced no evidence of irritation or sensitization. Moisturizing lotions containing 0.8% Myristyl Alcohol were nonirritating to human skin, and moisturizers containing 0.25% Myristyl Alcohol were neither irritants nor sensitizers. No signs of skin irritation or sensitization were observed in humans following the dermal application of 25% Isostearyl Alcohol. In a human skin sensitization study of a cream containing 3.0% Cetearyl Alcohol, none of the subjects had positive reactions. An analysis of the data and comparison with data from other toxicity studies on long-chain aliphatic alcohols is presented. Based on the available data included in this report, it is concluded that Cetearyl Alcohol, Cetyl Alcohol, Isostearyl Alcohol, Myristyl Alcohol, and Behenyl Alcohol are safe as cosmetic ingredients in the present practices of use.

7 Final Report on the Safety Assessment of Dibutyl Phthalate, Dimethyl Phthalate, and Diethyl Phthalate

Dibutyl Phthalate (DBP), Dimethyl Phthalate (DMP), and Diethyl Phthalate (DEP) are dialkyl phthalates used primarily in cosmetics at concentrations of less than 10 percent as plasticizers, solvents, and perfume fixatives. These phthalates are rapidly absorbed, metabolized, and excreted. Acute animal feeding studies indicate that these ingredients are nontoxic. The results of most subchronic and chronic tests indicate that these ingredients are relatively nontoxic to rats. The oral administration of DBP produced testicular atrophy in various test rodents. The available data are not adequate to prove that these ingredients are teratogenic agents to experimental animals. This was not observed after the administration of DMP and DEP. Undiluted DBP, DMP, and DEP produced only minimal irritation to eyes of rabbits. The mutagenic activity of DBP, DMP, and DEP toward Salmonella typhimurium mutants is essentially negative, but some assays reported positive findings. Carcinogenesis was not observed in DBP feeding studies. Limited clinical data on DBP, DMP, and DEP indicate that these ingredients are not human skin irritants, sensitizers, or phototoxic agents. On the basis of the available data, it is concluded that these compounds are safe for topical application in the present practices of use and concentration in cosmetics.

Final Report on the Safety Assessment of TEA-Lauryl Sulfate

TEA-Lauryl Sulfate is the triethanolamine salt of lauryl sulfuric acid. It is used in cosmetics as a detergent, a stabilizer and a solubilizer. The ingredient was moderately to slightly toxic in acute oral studies with rats; reported LD50s ranged from 0.27 to > 1.95 g/kg. Animal studies showed that the surfactant is a significant skin and eye irritant. In clinical studies, shampoos containing 10.5% TEA-Lauryl Sulfate caused no irritation under semioccluded conditions. Diluted shampoos containing 0.15-7.5% of the surfactant caused human skin reactions ranging from no irritation to moderate irritation. This skin irritation phenomenon is observed with most detergents. Undiluted shampoos containing 10.5% TEA-Lauryl Sulfate showed low potential for eliciting human skin sensitization. No evidence of photosensitization was observed in subjects exposed to solutions containing up to 0.42% TEA-Lauryl Sulfate. On the basis of the available animal and human data, the Panel concludes that TEA-Lauryl Sulfate can be used without significant irritation at a final concentration not exceeding 10.5%. Greater concentrations may cause irritation, especially if allowed to contact the skin for significant periods of time.

Cutaneous sensitivity to ultraviolet light and chemical irritants.

This investigation examines the relationship between the sun sensitivity of human skin and its response to chemical irritants. Forty-four Caucasoid subjects with normal back skin were studied. The minimal erythema dose (MED) was determined with the sunburning spectrum of a high-pressure mercury lamp. Cutaneous irritability was quantified using a series of seven irritants of different chemical structure, solubility, and concentrations. The response was either expressed as a threshold value of exposure time (ammonium hydroxide, sodium hydroxide) or was graded after a standard exposure in intensity of whealing (dimethyl sulphoxide) or erythema (sodium lauryl sulphate, quaternium 1, croton oil, kerosene). A significant correlation between the MED and the response to all seven primary irritants was found. The relationship was better for water-soluble irritants than for lipid-soluble ones. Despite marked individual variations the determination of the MED is suggested as a valuable tool in identifying hyperirritable skin. Skin typing based on complexion and sunburn history proved to be less reliable.

Usefulness and limitations of various guinea-pig test methods in detecting human skin sensitizers—Validation of guinea-pig tests for skin hypersensitivity

Several guinea-pig predictive test methods were evaluated by comparison of results with those obtained with human predictive tests, using ten compounds that have been used in cosmetics. The method involves the statistical analysis of the frequency with which guinea-pig tests agree with the findings of tests in humans. In addition, the frequencies of false positive and false negative predictive findings are considered and statistically analysed. The results clearly demonstrate the superiority of adjuvant tests (complete Freund's adjuvant) in determining skin sensitizers and the overall superiority of the guinea-pig maximization test in providing results similar to those obtained by human testing. A procedure is suggested for utilizing adjuvant and non-adjuvant test methods for characterizing compounds as of weak, moderate or strong sensitizing potential.

Skin sensitization potential of allyl methacrylate in guinea pigs.

The skin sensitization potential of allyl methacrylate (AMA) was studied in Hartley albino guinea pigs. Two groups of guinea pigs (10 per compound) were subjected to repeated insult patch tests either with AMA or with dinitrochlorobenzene (DNCB). All guinea pigs treated with DNCB (positive control) were sensitized. None of the guinea pigs treated with AMA exhibited any evidence of sensitization and no reactions attributable to AMA were elicited as a result of exposure during the insult phase of the patch test. Based on these results, it is concluded that allyl methacrylate is not likely to be a sensitizer and exposure to this material in industrial handling situations will not present an appreciable risk of human skin sensitization,

A collection of guinea pig sensitization test results--grouped by chemical class.

Various chemical groups were evaluated for their skin sensitization potential in the guinea pig. In general, amines, acetanilides, pyridines, piperidines, and sulfones were positive in the guinea pig test. Since these tests were done over a period of years, any further structural-related correlations or predictions should be made with caution due to variability of sample purity or differences in methodology. It is important to realize that every chemical which is positive in the guinea pig should not be construed as definitive evidence of human skin sensitization.

The sensitivity of human skin to UV-A as a function of time oral administration of 8-MOP.

The sensitivity of human skin to UV-A as a function of time oral administration of 8-MOP.

Incidence of human skin sensitization to isostearyl alcohol in two separate groups of panelists.

Human repeated insult patch testing with an experimental pump spray antiperspirant formulation elicited sensitization reactions in two separate groups of panelists. After several rechallenges with variations of the formula components, isostearyl alcohol at 5% concentration in the product emerged as the causative agent in both groups. Due to the high content of alcohol in the pump spray antiperspirant, control patch testing was done with 100% ethanol, which served as the vehicle for testing isostearyl alcohol alone. All reactions were negative. Six out of 148 participants in the first group and one of 60 in the second group exhibited sensitization responses to isostearyl alcohol.

Heat pain sensitivity of human skin after mild heat injury and its lack of dependence on the local blood flow

1. (1) Heat pain thresholds have been determined on the forearm of a group of inexperienced subjects using a radiant heat stimulator. During heating the skin temperature was estimated using a small thermocouple pressing on the skin. Linear rates of rise of skin temperature were produced by automatic feedback control of the heating lamp. 2. (2) Pain threshold was reached at an average temperature of 50.7°C when the rate of rise of temperature was 1.0°C/sec. With slower rates of heating the pain threshold was reached at a lower temperature. 3. (3) Heat pain thresholds fell by an average of 5.5°C following suprathreshold heating to 57.5°C. They did not fall significantly following heating to 55°C. 4. (4) Heat pain thresholds in hyperalgesic skin (and in normal skin) were unaffected when the blood, flow was arrested for up to 17 min by inflating a sphygmomanometer cuff around the upper arm. 5. (5) The lack of effect of stopping the blood flow is compared with other studies showing (a) similar results for heat injury of nociceptors in rabbit skin and (b) contrary findings for hyperalgesia following other types of tissue damage with slower onset in man.

Skin sensitization potential of trisodium ethylenediaminetetraacetate.

A sodium salt of ethylenediaminetetraacetate (Na3EDTA) and ethylenediamine (EDA) were subjected to a repeated insult patch test on Hartley albino guinea pigs (10 per compound). All guinea pigs (10 of 10) receiving EDA were sensitized. None of the guinea pigs (0 of 10) was sensitized to Na3 EDTA. Likewise, none of the guinea pigs sensitized to EDA reacted positively when challenged with Na3 EDTA. Based on these results, it is concluded that EDTA is not likely to be a sensitizer to humans, and would not likely cross-sensitize with EDA. In addition, the presence of very small amounts of the sodium salts of EDTA in cosmetic and pharmaceutical preparations does not represent an appreciable risk of human skin sensitization.

Mechanisms of reaginic hypersensitivity: a review.

SummaryGamma E antibodies represent the major part of human reaginic antibodies. The antibodies sensitize human and monkey skin, human leucocytes and primate lung tissues. The allergen‐γE antibody reaction mediates the release of histamine from human leucocytes and both histamine and SRS‐A from monkey lung tissues. The immunoglobulin is formed in lymphoid tissues especially those In respiratory and gastrointestinal tracts. Turnover rate of this protein in the blood is only 2–3 days, suggesting that human reaginic antibodies are being formed continuously in atopic patients. However, γE injected into normal skin sites is retained for a long time. The half‐time of loss of the immunoglobulin was 9–14 days in human skin and 8–10 days in monkey skin.Evidence has accumulated that IgE combines with target cells through the Fc portion of the molecules. The Fc fragments block passive sensitization of human skin with reaginic antibodies and passively sensitize human leucocytes and monkey lung tissue for reversed type reactions. Target cells in human leucocytes were identified as basophil leucocytes. The immunoglobulin binds with tissue mast cells upon passive sensitization.The initial step in reaginic hypersensitivity seems to be the bridging of cell‐bound IgE with allergen. It appears that the interaction between the IgE molecules or consequent structural changes in the Fc portion of the molecules may induce enzymatic sequences leading to the release of histamine and SRS‐A. The enzyme activated by the allergen‐γE antibody reaction is not the complement system. The biochemical pathways subsequent to the γE‐γE interaction and prior to the release of chemical mediators are unknown. However, evidence has accumulated that the β adrenergic system or the intracellular accumulation of cyclic‐AMP regulate the biochemical pathways.

Studies on IgE fragments prepared by papain digestion.

IgE (PS) myeloma protein was digested with papain for various lengths of time. After 1 h, several components of the IgE could already be demonstrated. After 3-11 h of digestion Fc, Fc′ fragments and a third component tentatively termed Fx were obtained. The Fc′ component showed partial identity with the Fc fragment, in the immunoelectrophoresis demonstrated faster mobility than the Fc fragment, had a molecular weight of approximately 58,000 and could not block passive sensitization of human skin by reaginic antibody. Thus far the Fx component was not obtained in a pure form. This component was demonstrated only in immunoelectrophoresis and had a slower mobility than the Fc and Fc′ fragments. For the time being it is not clear whether the Fx is a F(ab′)<sub>2</sub> fragment or a part of it, or is a third component of the Fc fragment.

Antigenic Structure of γE-Gobulin and Reaginic Antibody

Summary Both κ and λ-chain antigenic determinants were detected in γE-globulin by radioimmunoelectrophoresis. In addition, γE-globulin has antigenic determinants which are lacking in other immunoglobulins of the four known classes, i.e., γG, γA, γM and γD as well as in γA- and γG-subclasses. The protein did not contain the major antigenic determinants of the four known classes of immunoglobulins. The results indicate that γE-globulin represents a distinct class of immunoglobulin. Reaginic activity in atopic patients' sera was precipitated by anti-γE-antibody but not by either γM-anti-γM-antibody complexes or γG-antibody complexes containing γG2-, γG3- and γG4-globulins. The distribution of reaginic activity in chromatographic fractions did not parallel that of γG2- or γG3-globulin. Passive sensitization of human skin with reaginic antibody was not blocked by any of the myeloma proteins of known classes or subclasses, whereas a fraction containing γE-globulin had blocking activity. The results indicate that reaginic antibody activity is associated with γE-globulin.

Physicochemical Properties of Reaginic Antibody

Summary Serum fractions from ragweed sensitive patients were heated at 56°C for 2 to 4 hr and the effect of this treatment on the γG-, γA- and γE-antibodies against purified ragweed allergen (antigen E) was studied. After heating, both γG- and γA-antibodies showed radioactive precipitin bands in radioimmunodiffusion, whereas the γE radioactive band was lost. Disappearance of the radioactive γE band on heating is not due to loss of antigen-binding activity of γE-antibody but to loss of precipitability of the protein with anti-γE-antibody. By the heat-treatment, skinsensitizing γE-antibody was changed to blocking antibody. It was also found that a fraction containing γE-globulin nonspecifically blocked passive sensitization of human skin with reaginic antibody, and that the blocking activity of the preparation was lost by heating. The results indicate that the major reason for inactivation of skin sensitizing activity by heating is the loss of affinity of γE-antibody for human skin tissues rather than to destruction of the antibody-combinding sites of the molecule.

Use of the albino guinea-pig to detect the skin-sensitizing ability of chemicals.

The guinea-pig has been used for over 20 years to demonstrate skin-sensitizing ability in chemical compounds. Correlation between the results of workers in this field is difficult because of the wide range of conditions under which tests for sensitizing potential have been performed. This has made difficult any attempt to compare the relative abilities of various chemical compounds to produce skin sensitization. In a routine test for skin-sensitizing potential, solutions of suspected sensitizing substance have been applied over three days to the ears of guinea-pigs, and the flanks have been challenged one week later with a range of concentrations of suspected sensitizing substance. The erythematous reaction produced 24 hours after challenge was rated and compared with that in unsensitized controls. Various alternative methods of skin testing have been compared with this ear-flank test. The ear-flank test gives good, reproducible results with many classes of chemical compound, including types of compound not previously described as giving rise to sensitization in the guinea-pig or in man, and including some compounds which are known to have carcinogenic potential. It is also demonstrated that sensitizing potential is found more frequently among aromatic (aryl) than aliphatic (alkyl) compounds. Particularly strong sensitization reactions are produced by certain aryl halides, aryl isocyanates, aryl hydrazines, <i>N</i>-nitroso compounds, and aromatic nitroso-compounds. An attempt is made to relate the results of animal tests to reported cases of human skin sensitization.

Physicochemical properties of reaginic antibody: III. Further studies on the reaginic antibody in γA-globulin preparations

Three sera from ragweed-sensitive patients were fractionated by chromatography on DEAE cellulose columns and gel filtration. It was confirmed that the reaginic activity in the chromatographic fractions did not parallel γA-globulin concentration. The γA-globulin fractions from the same sera contained reaginic activity. When the γA-globulin preparations were further fractionated by chromatography on DEAE cellulosc columns, the reaginic activity was concentrated into one of the three subfractions. The precipitation of γA-globulin in the subfraction and the original γA-globulin preparation with rabbit anti-γA-globulin antibody was not accompanied by any detectable decrease of reaginic activity. The results indicated that the reaginic activity in the fractions was not associated with γA-globulin, but with impurities which were not detected by immunodiffusion methods. The subfractions of the γA-globulin preparation from a ragweed-sensitive patient blocked passive sensitization of normal human skin with reaginic antibody of a heterologous system. The blocking activity of the fractions paralleled the sensitizing activity against ragweed-pollen extract. It was also found that a fraction containing reagin but no detectable γA-globulin had a definite blocking activity, whereas γA-myeloma proteins did not. These findings suggest that both the blocking activity and reaginic activity are associated with the same protein other than γA-globulin.

Physicochemical properties of reaginic antibody: I. Association of reaginic activity with an immunoglobulin other than γA- or γG-globulin

Two reaginic sera from atopic patients were fractionated by chromatography on DEAE cellulose columns and by gel filtration through Sephadex G-200 columns. The skin-sensitizing activity in the chromatographic fractions did not parallel the concentration of either γA- or γG-globulin. Gamma G and γA-globulins in the reagin-containing fractions were precipitated with rabbit antibodies which are specific for either γG- or γA-globulin. Essentially all skin-sensitizing activity in the fractions remained in the supernatant after the precipitation of both immunoglobulins. The reaginic activity in the supernatant was coprecipitated with γG-globulin-anti-light chain antibody complexes. These results indicated that the skin-sensitizing antibody was associated with neither γG- nor γA-globulin but with a unique protein having light chain(s) of immunoglobulins. It was also found that 0.001 to 0.002 μg N of this protein was sufficient for the passive sensitization of normal human skin for positive P-K reactions. The possible carrier of reaginic activity was discussed.

Effect of reduction and alkylation on allergen-combining properties of reaginic antibody

The γA-globulin fraction of a reaginic serum was reduced in 0.1M mercaptoethanol followed by alkylation with iodoacetate. Reaginic antibody in the preparation was inactivated by the treatment. The reduced-alkylated γA-globulin did not manifest blocking antibody activity in the Prausnitz-Küstner reaction when it was incubated with allergen. Pretreatment of allergen-coated polyaminostyrene with untreated reaginic γA-globulin decreased the activity of the immune adsorbent to combine with the reagin, whereas treatment with the reduced-alkylated sample did not, indicating that the allergen-combining activity of the reagin was greatly diminished by the reduction-alkylation treatment. The reduced-alkylated γA-globulin from either normal or reaginic serum partially blocked passive sensitization of normal human skin with reagin, suggesting that affinity of reaginic antibody for tissues remained after the treatment. The results definitely indicate that the allergen-combining site(s) in reaginic antibody in the γA-globulin fraction is degraded by the reduction-alkylation treatment.