Human systemic exposure to [¹4C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment.

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [(14)C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [(14)C] was measured in all materials, rinsing water, hair, plasma, urine and skin strips. Plasma and urine were also analysed by HLPC/MS/MS for PPD and its metabolites (B). Total mean recovery of radioactivity was 94.30% (A) or 96.21% (B). Mean plasma Cmax values were 132.6 or 97.4 ng [(14)C]-PPDeq/mL, mean AUC(0-∞) values 1415 or 966 ng [(14)C]-PPDeq/mL*hr in studies A or B, respectively. Urinary excretion of [(14)C] mainly occurred within 24 hrs after hair colouring with a total excretion of 0.72 or 0.88% of applied radioactivity in studies A or B, respectively. Only N,N'-diacetylated-PPD was detected in plasma and the urine. A TK-based human safety assessment estimated margins of safety of 23.3- or 65-fold relative to respective plasma AUC or Cmax values in rats at the NOAEL of a toxicity study. Overall, hair dyes containing PPD are unlikely to pose a health risk since they are used intermittently and systemic exposure is limited to the detoxified metabolite N,N'-diacetyl-PPD.

Safety of botanical ingredients in personal care products/cosmetics.

The key issue of the safety assessment of botanical ingredients in personal care products (PCP) is the phytochemical characterisation of the plant source, data on contamination, adulteration and hazardous residues. The comparative approach used in the safety assessment of GM-plants may be applied to novel botanical PCP ingredients. Comparator(s) are the parent plant or varieties of the same species. Chemical grouping includes definition of chemical groups suitable for a read-across approach; it allows the estimation of toxicological endpoints on the basis of data from related substances (congeneric groups) with physical/chemical properties producing similar toxicities. The Threshold of Toxicological Concern (TTC) and Dermal Sensitisation Threshold (DST) are tools for the assessment of trace substances or minor ingredients. The evaluation of skin penetration of substances present in human food is unnecessary, whereas mixtures may be assessed on the basis of physical/chemical properties of individual substances. Adverse dermal effects of botanicals include irritation, sensitisation, phototoxicity and immediate-type allergy. The experience from dietary supplements or herbal medicines showed that being natural is not equivalent to being safe. Pragmatic approaches for quality and safety standards of botanical ingredients are needed; consumer safety should be the first objective of conventional and botanical PCP ingredients.

Safety assessment of personal care products/cosmetics and their ingredients.

We attempt to review the safety assessment of personal care products (PCP) and ingredients that are representative and pose complex safety issues. PCP are generally applied to human skin and mainly produce local exposure, although skin penetration or use in the oral cavity, on the face, lips, eyes and mucosa may also produce human systemic exposure. In the EU, US and Japan, the safety of PCP is regulated under cosmetic and/or drug regulations. Oxidative hair dyes contain arylamines, the most chemically reactive ingredients of PCP. Although arylamines have an allergic potential, taking into account the high number of consumers exposed, the incidence and prevalence of hair dye allergy appears to be low and stable. A recent (2001) epidemiology study suggested an association of oxidative hair dye use and increased bladder cancer risk in consumers, although this was not confirmed by subsequent or previous epidemiologic investigations. The results of genetic toxicity, carcinogenicity and reproductive toxicity studies suggest that modern hair dyes and their ingredients pose no genotoxic, carcinogenic or reproductive risk. Recent reports suggest that arylamines contained in oxidative hair dyes are N-acetylated in human or mammalian skin resulting in systemic exposure to traces of detoxified, i.e. non-genotoxic, metabolites, whereas human hepatocytes were unable to transform hair dye arylamines to potentially carcinogenic metabolites. An expert panel of the International Agency on Research of Cancer (IARC) concluded that there is no evidence for a causal association of hair dye exposure with an elevated cancer risk in consumers. Ultraviolet filters have important benefits by protecting the consumer against adverse effects of UV radiation; these substances undergo a stringent safety evaluation under current international regulations prior to their marketing. Concerns were also raised about the safety of solid nanoparticles in PCP, mainly TiO(2) and ZnO in sunscreens. However, current evidence suggests that these particles are non-toxic, do not penetrate into or through normal or compromised human skin and, therefore, pose no risk to human health. The increasing use of natural plant ingredients in personal care products raised new safety issues that require novel approaches to their safety evaluation similar to those of plant-derived food ingredients. For example, the Threshold of Toxicological Concern (TTC) is a promising tool to assess the safety of substances present at trace levels as well as minor ingredients of plant-derived substances. The potential human systemic exposure to PCP ingredients is increasingly estimated on the basis of in vitro skin penetration data. However, new evidence suggests that the in vitro test may overestimate human systemic exposure to PCP ingredients due to the absence of metabolism in cadaver skin or misclassification of skin residues that, in vivo, remain in the stratum corneum or hair follicle openings, i.e. outside the living skin. Overall, today's safety assessment of PCP and their ingredients is not only based on science, but also on their respective regulatory status as well as other issues, such as the ethics of animal testing. Nevertheless, the record shows that today's PCP are safe and offer multiple benefits to quality of life and health of the consumer. In the interest of all stakeholders, consumers, regulatory bodies and producers, there is an urgent need for an international harmonization on the status and safety requirements of these products and their ingredients.

Retinyl palmitate is non-genotoxic in Chinese hamster ovary cells in the dark or after pre-irradiation or simultaneous irradiation with UV light.

Retinyl palmitate (RP), an ingredient of cosmetic and medical skin-care preparations, has been reported to be photo-genotoxic/photo-clastogenic in mouse lymphoma cells (Tk locus) as well as in human Jurkat T-cells, as measured by use of the comet assay. Given that these results were obtained under exploratory conditions, we re-investigated the photo-genotoxicity of RP following a protocol consistent with current recommendations for photo-genotoxicity testing of drugs and chemicals. We tested RP in Chinese hamster ovary (CHO) cells in the dark (standard chromosome aberration test), under pre-irradiation (UVA irradiation of cells and subsequent treatment with RP) or simultaneous irradiation (irradiation of cells and RP together, standard photo-genotoxicity protocol) conditions. UVA irradiation was applied at 350 and 700 mJ/cm2 with the high UV dose targeted to produce a small increase in the incidence of structural chromosome aberrations (CA) in cells not treated with RP. RP was tested up to and above its limit of solubility in the culture medium (20-40 µg/mL). There was no overt cytotoxicity under dark or different irradiation conditions. Treatment of cells with RP in the dark, as well as treatment under pre- or simultaneous irradiation conditions failed to produce biologically significant increases in the incidence of CA, whereas the positive control substances 4-nitroquinolone and 8-methoxypsoralene produced significantly positive effects in the dark or under simultaneous irradiation, respectively. Overall, our results failed to confirm the reported positive photo-genotoxic effects, and suggest that they may have been due to the test conditions, i.e. high irradiation doses, high cytotoxicity or re-irradiation of photo-products. In conclusion, our data suggest that, under standard conditions for testing photo-genotoxicity, RP had no in vitro genotoxic or photo-genotoxic potential and is therefore unlikely to pose a local or systemic genotoxic or photo-genotoxic risk.

Occupational exposure of hairdressers to [14C]-para-phenylenediamine-containing oxidative hair dyes: a mass balance study.

We monitored the exposure of hairdressers to oxidative hair dyes for 6 working days under controlled conditions. Eighteen professional hairdressers (3/day) coloured hairdresser's training heads bearing natural human hair (hair length: approximately 30 cm) for 6 h/working day with a dark-shade oxidative hair dye containing 2% [14C]-para-phenylenediamine (PPD). Three separate phases of hair dyeing were monitored: (A) dye preparation/hair dyeing, (B) rinsing/shampooing/conditioning and (C) cutting/drying/styling. Ambient air and personal monitoring samples (vapours and particles), nasal and hand rinses were collected during all study phases. Urine (pre-exposure, quantitative samples for the 0-12, 12-24, 24-48 h periods after start of exposure) and blood samples (blank, 4, 8 or 24 h) were collected from all exposed subjects. Radioactivity was determined in all biological samples and study materials, tools and washing liquids, and a [14C]-mass balance was performed daily. No adverse events were noted during the study. Waste, equipment, gloves and coveralls contained 0.41+/-0.16%, dye mixing bowls 2.88+/-0.54%, hair wash 45.47+/-2.95%, hair+scalp 53.46+/-4.06% of the applied radioactivity, respectively. Plasma levels were below the limit of quantification (10 ng PPDeq/mL). Total urinary 0-48 h excretion of [14C] levels ranged from a total of <2-18 microg PPDeq and was similar in subjects exposed during the different phases of hair dyeing. Minimal air levels at or slightly above the limit of quantification were found in a few personal air monitoring samples during the phases of hair dyeing and hair cutting, but not during the rinsing phase. Air area monitoring samples or nasal rinses contained no measurable radioactivity. Hand residues ranged from 0.006 to 0.15 microg PPDeq/cm2, and were found predominantly after the cutting/drying phase. The mean mass balance of [14C] across the six study days was 102.50+/-2.20%. Overall, the mean, total systemic exposure of hairdressers to oxidative hair dyes during a working day including 6 hair dyeing processes was estimated to be <0.36 microg PPDeq/kg body weight/working day. Our results suggest that (a) current safety precautions for the handling of hair dyes offer sufficient protection against local and systemic exposure and (b) professional exposure to oxidative hair dyes does not pose a risk to human health.

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites.

para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells.

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.

Under the skin: Biotransformation of para-aminophenol and para-phenylenediamine in reconstructed human epidermis and human hepatocytes.

We investigated the biotransformation of the oxidative arylamine (AA) hair dye ingredients [14C]-para-aminophenol (PAP) and [14C]-para-phenylenediamine (PPD) in reconstructed human epidermis and human hepatocytes. Human epidermis quantitatively transformed PAP to its N-acetylated derivative (APAP), whereas hepatocytes transformed PAP to sulfate or glucuronic acid conjugates of APAP or PAP as well as free APAP. Epidermis and hepatocytes converted PPD to N-mono- (MAPPD) and N,N'-di-acetylated (DAPPD) derivatives. At higher concentrations of PPD (250-1000 microM), epidermis or hepatocytes produced more of the MAPPD, whereas concentrations below 250 microM and lower favoured formation of the DAPPD metabolite. When compared with epidermis, human hepatocytes had a three-fold or eight-fold greater capacity for generation of MAPPD or DAPPD, respectively. No evidence of transformation of PAP or PPD to N-hydroxylated derivatives was found in epidermis or hepatocytes. Our results suggest that (i) after dermal absorption of PAP or PPD, humans are systemically exposed to acetylated derivatives; (ii) current in vitro skin absorption studies may be inadapated for determination of human systemic exposure to AAs due to reduced or absent metabolic capacity of non-viable skin; (iii) due to qualitative differences between dermal and hepatic metabolism, oral toxicity studies may be unsuited for the hazard assessment of dermal exposure to AAs; and (iv) use of induced rodent liver S9 metabolic activation systems for in vitro genotoxicity studies may produce misleading results on the hazard of human dermal exposure to AAs. In conclusion, our data support the growing evidence that AAs are transformed in human skin and suggest that current practices of safety assessment of AAs should take these findings into account.

Sensitivity and specificity of the consumer open skin allergy test as a method of prediction of contact dermatitis to hair dyes.

To prevent contact dermatitis to oxidative hair colouring products, a consumer test (skin allergy test, SAT) consisting of the open application of the colourant base prior to mixing with the developer is recommended 48 hours before hair colouring. We investigated the sensitivity and specificity of the SAT to detect and prevent contact allergy to oxidative hair colouring products that contained a range of concentrations of para-phenylenediamine (PPD) and corresponded to different shades (light, medium and dark). Test colouring products containing increasing concentrations of PPD (0.1, 0.5, 1.0 or 1.5%) were applied to 34 PPD-positive hair dye-allergic individuals and to 49 non-allergic control subjects. Allergic reactions were elicited in all PPD-positive subjects whereas none occurred in control PPD-negative subjects. For each subject the eliciting concentration of PPD in the SAT was compared with the PPD concentration range of the group of commercial shades reported as causing reactions by the consumer. In all PPD-positive subjects the eliciting concentrations of PPD in the SAT was within or lower than the range of PPD concentrations in the reported eliciting colourant base of commercial products. In conclusion, our results confirm the excellent predictive value of the SAT over the entire range of PPD concentrations used in oxidative hair colouring products and suggest that the test is a suitable tool for the secondary prevention of contact allergic reactions to hair colouring products.

Urinary acetylated metabolites and N-acetyltransferase-2 genotype in human subjects treated with a para-phenylenediamine-containing oxidative hair dye.

In the organism of mammals, important detoxification pathways of arylamines are catalysed by N-acetyltransferase 2 (NAT2). A recent case-control epidemiology study suggested that human NAT2 slow acetylators exposed to oxidative hair dyes may be at greater risk to develop bladder cancer. We therefore profiled urinary [(14)C]-metabolites and NAT2 genotype in eight human subjects following treatment with a dark-shade oxidative hair dye containing [(14)C]-para-phenylenediamine (PPD). Genotyping identified three subjects as slow, and five subjects as intermediate NAT2 acetylators. Within 24 h after treatment, the study subjects excreted a mean total of 0.43+/-0.24% of the applied [(14)C] in the urine, where five different metabolites were found. The major urinary metabolites were concluded to be N-mono-acetylated and N,N'-diacetylated PPD. They were present in all urine samples and amounted to 80-95% of the total urinary [(14)C]. Another metabolite, possibly a glucuronic acid conjugate, was found in 6/8 urine samples at 5-13% of the total urinary [(14)C]. All metabolites appeared to be related to PPD, no evidence of the presence of high-molecular weight dye-intermediates or corresponding metabolites was found. The metabolite profile in the study subjects showed no significant differences between the NAT2 intermediate and NAT2 slow acetylator subgroups. Urine of NAT2 slow acetylators contained N-mono-acetylated-PPD at 42.2+/-10.2% and N,N'-di-acetylated-PPD at 54.1+/-7.6% of total urinary radioactivity, while the corresponding values of intermediate acetylators were 46.0+/-8.9% and 45.7+/-9.9%, respectively. Overall, our results suggest that the human acetylation rate of PPD after topical application is independent of the NAT2 genotype status, most likely due to metabolism by epidermal NAT1 prior to systemic absorption.

Human systemic exposure to a [14C]-para-phenylenediamine-containing oxidative hair dye and correlation with in vitro percutaneous absorption in human or pig skin.

We investigated the absorption of a commercial [14C]-PPD-containing oxidative dark-shade hair dye in human volunteers as well as in vitro using human or pig ear skin. The hair of eight male volunteers was cut to a standard length, dyed, washed, dried, clipped and collected. Hair, washing water, materials used in the study and a 24-h scalp wash were collected for determination of radioactivity. Blood, urine and faeces were analysed up to 120 h after hair dyeing. An identical [14C]-PPD-containing hair dye formulation was applied in vitro for 0.5 h to human and pig ear skin, and radioactivity was determined in skin compartments after 24 h. In humans, the recovery rate was 95.7+/-1.5% of the applied radioactivity. Washing water, cut hair, gloves, paper towels, caps or scalp wash contained a total of 95.16+/-1.46% of the applied [14C]. Absorbed radioactivity amounted to 0.50+/-0.24% in the urine and 0.04+/-0.04% in the faeces, corresponding to a mean of 7.0+/-3.4 mg [14C]-PPD-equivalents absorbed. Within 24 h after application, most of the radioactivity was eliminated. The Cmax of [14C]-PPD-equivalents in the plasma was 0.087 microgeq/ml, the Tmax was approximately 2 h, and the mean the AUC(0-12h) was 0.67 microgeq h/ml. In vitro tests in human or pig skin found total absorbed amounts of 2.4+/-1.6% (10.6+/-6.7 microgeq/cm2) or 3.4+/-1.7% (14.6+/-6.9 microgeq/cm2), respectively. Percentage-based in vitro results were considerably higher than corresponding in vivo data, whereas, in units of microg/cm2, they corresponded to a total absorbed amount of 7.40 or 10.22 mgeq for human or pig skin, respectively. All results suggested that hair dyeing with oxidative hair dyes produces minimal systemic exposure that is unlikely to pose a risk to human health.

Toxicity and human health risk of hair dyes.

Hair dyes and their ingredients have moderate to low acute toxicity. Human poisoning accidents are rare and have only been reported following oral ingestion. Contact sensitisation to hair dyes has been a safety issue, mainly as a consequence of unprotected professional exposure. Although the use of hair dyes has dramatically increased in industrialised countries during the last decades, the prevalence of sensitisation to hair dyes in the general and professional populations has stabilised or declined. In vitro genotoxicity tests on hair dye ingredients frequently had positive results, although their correlation with in vivo carcinogenicity for the chemical class of oxidative hair dye ingredients (aromatic amines) is uncertain. Positive in vivo genotoxicity results on hair dyes are rare. Studies in man found no evidence of genotoxic effects of hair dyes or their ingredients. On the basis of mechanistic studies, some in vivo positive hair dye ingredients (p-aminophenol, Lawsone) have been shown to pose no or negligible risk to human health. Although a recent case-control epidemiology study suggested an association of hair dye use and bladder cancer, a number of other studies, including prospective investigations on large populations, found no or negative correlations for bladder or other cancers. Although in vivo topical carcinogenicity studies on hair dye ingredients or commercial formulations yielded no evidence for systemic toxicity or carcinogenicity, oral carcinogenicity studies on hair dye ingredients at oral doses up to the maximum tolerated dose (MTD) suggested that some ingredients are carcinogenic in rodents. Human systemic exposure to various (14)C-labelled oxidative hair dyes under conditions of use was below 1.0% of the amount applied. Conservative risk assessments suggested no or negligible cancer risk, including for ingredients that were found to be positive in oral carcinogenicity studies. The results of reproductive toxicity studies and epidemiological investigations suggested that hair dyes and their ingredients pose no risk of adverse reproductive effects. In conclusion, the weight of evidence suggests that consumer or professional exposure to hair dyes poses no carcinogenic or other human health risks.

An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one].

Kojic acid (KA), a natural substance produced by fungi or bacteria, such as Aspergillus, Penicillium or Acetobacter spp, is contained in traditional Japanese fermented foods and is used as a dermatological skin-lightening agent. High concentrations of KA (>or=1000 microg/plate) were mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA102 and E. coli WP2uvrA, but not in TA 1537. An Ames test following the "treat and plate" protocol was negative. A chromosome aberration test in V79 cells following a robust protocol showed only a marginal increase in chromosome aberrations at cytotoxic concentrations after prolonged (>or=18 h) exposure. No genotoxic activity was observed for hprt mutations either in mouse lymphoma or V79 cells, or in in vitro micronucleus tests in human keratinocytes or hepatocytes. All in vivo genotoxicity studies on KA doses were negative, including mouse bone marrow micronucleus tests after single or multiple doses, an in vivo/in vitro unscheduled DNA synthesis (UDS) test, or a study in the liver of the transgenic Muta(TM) Mouse. On the basis of pharmacokinetic studies in rats and in vitro absorption studies in human skin, the systemic exposure of KA in man following its topical application is estimated to be in the range of 0.03-0.06 mg/kg/day. Comparing these values with the NOAEL in oral subchronic animal studies (250 mg/kg/day), the calculated margin of safety would be 4200- to 8900-fold. Comparing human exposure with the doses that were negative for micronuclei, UDS and gene mutations in vivo, the margins of safety are 16000 to 26000-fold. In conclusion, the topical use of KA as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.

Contact sensitivity to hair dyes can be detected by the consumer open test.

To avoid allergic reactions to hair dyes, an open test ("skin sensitivity test" or "dab test") is recommended 48 hours before the hair colouring procedure. We have investigated in a multicenter study, under standardized conditions and medical supervision, the validity of this test as a practical method to detect allergy to paraphenylenediamine (PPD)-containing hair dyes in 30 PPD patch test-positive and 30 PPD patch test-negative subjects. A defined volume of a marketed hair colouring product containing 1.8% PPD was applied in an open patch test to the retroauricular area. The grading method comprised the dermatologist's clinical evaluation and objective numerical scores. Allergic reactions were elicited in all PPD-positive subjects with a maximal intensity on Day 2; no allergic reactions were elicited in PPD-negative subjects. Increased severity of reactions by the dermatologist's clinical evaluation was correlated strongly with increasing numerical scores. The "skin sensitivity test" can be considered as an effective method to detect type IV hair dye allergy and as such, as an important factor in its secondary prevention.