Seven volunteers (6 females and 1 male, age 19-60 years, median age 27 years) were enrolled in the study. They had been previously tested with 2,5-TDA-S 1% in petrolatum (pet.) and PPD 1% in pet. at the Department of Occupational and Environmental Dermatology (DOED) in Malmo, and presented with a ++ or +++ reactions according to the International Contact Dermatitis Research Group (ICDRG) criteria 89. Exclusion criteria were sensitization to fragrances and rubber derivatives. The participants were recruited by phone call. The subjects were sent written detailed information on the study before inclusion.

Two nitrile gloves of different brands and one vinyl glove were investigated (Table 1). The gloves provided in the package of the at-home hair dye were investigated with the same dye in all the 7 volunteers and in 5 of them also using 3 professional hair dyes. As negative control the 4H^®-glove (made of layers of polyethylene/ethylene-vinyl-alcohol laminate) (North Safety Products by Honeywell, Smithfield, USA) with known protective ability against chemicals 10, was used.

Three permanent hair dyes (Table 2) intended for professional use (HDp), 1 containing PPD (HDp-PPD), 1 containing 2,5 TDA sulphate (HDp-TDA-S), 1 containing ME-PPD (HDp-ME-PPD), and 1 hair dye for home use (HDhu) and containing 2,5-TDA-S (HDhu-TDA-S), were used. The hair dyes tested were prepared, simulating their ordinary use, by mixing the colouring cream with the corresponding developer containing hydrogen peroxide 3% (10 vol.) or 6% (20 vol.) with a mixing ratio of 1:1 w:w, immediately before the application in the test chambers system.

Fourier-transform infrared (FTIR) spectroscopy was performed with an Agilent Cary 630 FTIR spectrometer (Agilent Technologies, Danbury, CT, USA) was used to analyse the material of the gloves supplied with the hair dye for the home use and labelled as plastic on the package leaflets. The method compared the spectrum of the analysed material with a database of the spectra of a wide range of materials. The thickness of gloves was measured at the palm/back of three samples for each glove by using a micrometer screw-gauge (Table 1).

High performance liquid chromatography (HPLC) analysis of the hair dyes was performed according to the method described by Antelmi et al. in 2016 10, detecting the PPD and 2,5-TDA content in all the colouring creams (before adding hydrogen peroxide) (Table 2). The analysis with regard to PPD and 2,5-TDA content was performed also in the ME-PPD-containing hair dye, the two chemicals were not detected given the quantification limit of 0.05% w/w. The ME-PPD content of the hair dye was not analysed since the reference substance could not be purchased at the time.

The exposure time for the provocation test was chosen based on information acquired in workplace visits to hairdressers saloons in Malmo. A maximum time of 45 minutes wearing gloves was thought appropriate. With regard to the at-home kit to dye hair, the instructions for use provided by the leaflet of the hair dye were used and 30 minutes was estimated as exposure time while wearing the gloves provided in the package, considering 5-10 minutes for the colouring procedure at-home and 5-20 minutes for the following wash of the newly dyed hair (no more than one pair of gloves is offered in each package).

To establish the current reactivity of the volunteers, a dilution series of PPD in ethanol (w/v) and 2,5-TDA-S in pet. (w/w) (1.0%, 0.1%, 0.01%, 0.001%, 0.0001%) were patch tested. For those with a previous +++ reaction to PPD and/or 2,5-TDA-S, the highest concentration tested was fixed at 10 times less than the threshold of reactivity previously observed in each subject. Finn Chambers^® (Ø8 mm) (Smart Practice, Phoenix, AZ, USA) on Scanpor^® tape (Norgesplaster A/S Vennesla, Norway) were used for patch testing the dilution series. 15 µl of  test solution, applied with a micropipette, for the PPD dilution series and 20 mg of pet. for the 2,5-TDA-S dilution series respectively were applied in each test chamber. The chambers were put on the back of the patients at the same time as the in vivo study and left for 2 days (48 hours), the reading was performed at day 4 (D4) and D7. The reactions were scored according to the ICDRG criteria 89.

In Vivo Provocation Test

The in vivo provocation test was carried out with an open chamber test system (Figure 1) developed at the DOED in Malmo to evaluate in vivo the permeation through gloves of different chemicals 610. The procedure of testing has been described elsewhere 610. During the provocation the subject lies in the supine position exposing the back. The chambers with the substance to be tested are applied to the back for fixed periods of time. The procedure is carried out with standardised humidity and temperature. The chambers are prepared with the glove material to be tested fixed with metal clips the bottom of the chamber ie where the chamber is fixed to the skin of back, thus acting as a membrane between hairdye and skin, simulating the actual effect of a glove in the same material (Figure 1,Figure 2).

In the study, the hair dye mixtures (prepared to simulate ordinary use, see above) was prepared within 4 minutes prior to the application. The professional hair dyes were tested with the three gloves (2 nitrile, 1 polyvinyl chloride), a negative control consisting of 4H^® glove, and a positive control consisting of a chamber devoid of glove on the bottom, filled with the hair dye directly in contact with the skin. The exposure time chosen was 45 minutes. For the home-use hair dye the polyethylene glove supplied in the package was tested at the exposure time of 30 minutes. The chambers were filled until half height (approximately 6 mm) with the hair dye/peroxide mixture (Figure 2) after removal of the test devices, a clinical examination of the skin corresponding to the test application area, was performed to detect the presence of dark spots as visible signs of permeation through the gloves. The integrity of glove material  tested and the possible presence of breakages were verified by cleansing the glove material and through visual examination. Readings were performed of the areas according to the ICDRG guidelines on D4 and D7 89. The test procedures and test readings were photo-documented.

The subjects gave informed consent and the Regional Ethical Review Board in Lund, Sweden, approved the protocol. The study was conducted in accordance with ethical standards on human experimentation and with the Helsinki Declaration of 1975, as revised in 1983.

McNemar s test two-sided was used for pair-wise comparison of the protective effect of the four gloves (2 nitrile, 1 polyvinyl chloride, 1 polyethylene) and of the test performed without the glove as membrane between the chambers/hair dyes and the skin (positive control). One-sided McNemar s test was used to compare the protective effect of each glove and the positive control, versus the 4H^® glove (negative control). A p-value below 0.05 was considered significant.For the same glove pair-wise comparisons using Fisher s test were made to compare the protective effects against the four hair dyes.

The gloves supplied in the hair dye for home use was analysed with Agilent Cary 630 FTIR spectrometer and it was found that the material was polyethylene. The average thickness of the gloves measured with a micrometer screw-gauge was within a range of 0.03- 0.13 mm (Table 1).

All the volunteers were tested with the open chamber test without any glove membrane (positive control) for 45 min of exposure with the 3 HDp, (HDp-TDA-S, HDp-PPD, HDp-ME-PPD) and for 30 min with the HDhu-TDA-S and all  developed positive reactions except for two who did not react to the test with HDp-ME-PPD (Figure 3, Table 3).

With the open chamber test with the 4H^® glove (negative control) and when the 4 hair dyes were tested with the 2 nitrile gloves  there were no positive.

The polyvinyl chloride glove did not give any protection in 6 volunteers when used with the hair dye HDp-TDA-S, and neither in 5 volunteers when used with HDp-PPD and HDhu-TDA- S. Regarding the hair dye HDp-ME-PPD however no  reactions were noted with the polyvinyl chloride glove (Table 3). Statistically, the 4H^® glove gave better protection than the polyvinyl chloride glove for all hair dyes except HDp-ME-PPD (Table 4). There was no difference between the nitrile gloves and 4H^®. The 2 nitrile gloves gave b etter protection than the polyvinyl chloride against the HDp-TDA-S in 6 subject, results being statistically significant (p= 0.031); between the HDp-PPD and the HDhu-TDA-S the difference was not significant however with a result close to statistically so  (p=0.062). No difference in term of reactions detected on the skin at test reading 1 (D4) and 2 (D7) was found between the nitrile and polyvinyl chloride gloves tested with HDp-ME-PPD.

The polyethylene glove protected all 7 tested individuals exposed for 30 min to the HDhu-TDA-S, and all the 5 individuals tested with HDp-ME-PPD for 45 min. With this longer exposure time, there was no protection obtained neither with the polyethylene glove concerning 4 of  the 5 tested individuals with HDp-TDA-S nor for 2 of the 5 volunteers tested with HDp-PPD (Table 3). Comparing the negative control with the polyethylene glove concerning the protection against the HDp-TDA-S, a statistical significance was indicated (p= 0.062) (Table 4). The 4H^® glove and the polyethylene glove gave the same protection with HDhu-TDA-S.

When tested with the HDhu-TDA-S hair dye for 30 minutes, the protection of polyvinyl chloride was adequate only for  2 subjects of 7. In the small group of 5 subjects tested with the polyethylene gloves and professional hair dyes at 45 minutes, results were not statistically significant but the trend comparing polyethylene and polyvinyl chloride gloves was the same when they were tested with HDp-ME-PPD and with HDp-TDA-S (Table 3).

Thus, the 4H^® and nitrile gloves gave complete protection regardless of what hair dye was used.  For the polyethylene and polyvinyl chloride gloves, respectively, complete protection was obtained against the HDp-ME-PPD but not against the other three hair dyes (Table 3, Figure 3).

No association was found for the polyvinyl chloride glove concerning the individual degree of patch test reactivity to PPD and the protective capacity, whereas with regard to 2,5-TDA-S reactivity, a numerical but not statistical association was indicated.

In a profession such as the hairdressers where there is a huge  exposure to both irritants and potentially contact allergic chemicals, the choice of gloves is of course important, and if advices can be based on evaluation simulating real life this is an obvious advantage. A method to  evaluate the performance of glove material could thus be through  in vivo studies mimicking the workplace exposure. In order to improve the advice given to hairdressers regarding protective gloves, we performed a study a few years ago 6 comparing the protective capacity of glove materials commonly used among hairdressers when exposed to a hair dye containing PPD. A good protection from nitrile gloves was noted. An interventional workplace-based study 11 concluded that when used correct, gloves can be protective in real work situations. Geens et al. 11 investigated the systemic exposure of hairdressers to 2,5-TDA-S and PPD using urine diamine biomarkers before and after ordinary work with permanent hair dyes the first week and again after providing nitrile gloves to hairdressers in the second week. Through the biomonitoring of PPD and 2,5-TDA-S a significant decrease of systemic exposure to 2,5-TDA-S but not to PPD was noted explained by the adequate use of nitrile gloves.

With regard to the exposure to hair dye ingredients, they vary qualitatively and quantitatively in different regions of the world. It is known that hair dyes in northern Europe more often contain 2,5-TDA or 2,5-TDA-S than PPD, or these dye ingredients combined 121314.  Furthermore, products bought in non-European countries usually contain higher concentrations of PPD 12. When comparing patch test results from dermatitis patients tested between 2003 and 2007 in 10 European patch test centres were compared, PPD sensitization occurred more often in Central and Southern Europe than in Scandinavia 15. The difference was thought to be due to the use of hair dyes with a darker shades more frequently. Moreover, a new hair dye substance, ME-PPD, was recently introduced in Europe 161718,  with  the aim to decrease the risk of sensitization and elicitation of allergic contact dermatitis.

In this study we wanted to further investigate the nitrile gloves that have already been found superior with regard to PPD 6 to evaluate them and other commonly used gloves for professional use and for home-dyeing, when using a PPD-, a 2,5-TDA-S- and a ME-PPD-containing hair dye for professional use and a 2,5-TDA-S-containing hair dye for home use. In order to get information on the hair dye products recommended for hairdressers during education and to collect information on how and to what degree protective measurements were taught, a visit in a school for apprentices hairdressers in Malmo was performed. Information on choice of gloves were collected also by performing a short interview with professional hairdressers, visiting 11 hairdressers saloons in the city of Malmo. The information was used to improve the actual study with regard to material used and time of exposure. In the study, where the aim was to evaluate gloves and hair dyes, a fixed time was decided. According to the outcome of our preliminary investigation including both hairdresser apprentices and professional hairdressers, we evaluated that a maximum exposure of 45 minutes in professional use and 30 minutes in the at-home dye condition was plausible.  

The performance of the polyethylene glove supplied in the package of a home-use hair dye was completely protective in all the subjects against the home-use hair dye, at 30 minutes of exposure, according to the instruction in the package. When the same glove was tested with professional hair dyes only (in 5 of the 7 individuals), its performance was poor most probably due to the increased exposure time (45 min).

Comparing the protection offered by the polyvinyl chloride gloves when using the different professional hair dyes, these gloves did not have a good performance  since 5 and 6 of 7 subjects reacted when tested with the HDp-PPD and with HDp-TDA-S respectively, and 5 of 7 subjects reacted at 30 minutes of exposure with HDhu-TDA-S.

With regard to polyvinyl chloride (tested in 7 subjects) and polyethylene (tested in 5 subjects)  with the HDp-ME-PPD, excellent protection was shown. On the other hand considering that the hair dye containing ME-PPD has recently been introduced on the market, we do not really know which reactivity pattern patients primarily sensitized to the ME-PPD would have. Therefore a larger group of individuals should be tested to verify if the good protection provided by the  gloves tested with this hair dye is due to a lower allergenic potential of the hair dye-ME-PPD-based. Previous patch testing with ME-PPD showed that there is decreased strength of elicitation response to this hair dye containing ME-PPD in PPD/TDA allergic subjects 17 and it has been shown that the hair dye products containing ME-PPD were tolerated by 29/43 PPD/TDA allergic individuals throughout continued hair dyeing with repeated treatments 18. In our study we could not patch test with dilution series of the substance as such and thus we have no knowledge of the actual reactivity pattern with regard to ME-PPD.  The two nitrile gloves gave complete protection with the three professional hair dyes at 45 minutes and with the home use hair dye at 30 minutes, comparable to the 4H^® glove (Figure 3, Figure 4, Figure 5), known to have excellent performance with regard to protection against chemicals 610.

In term of advice to hairdressers the study has shown that the nitrile glove must be the preferred choice during the hair dye procedures with the permanent hair dyes currently available. Also with regard to ME-PPD, nitrile gloves is easiest to advice since we know that these will protect the consumer. However limited, the study indicates that the use of hair dye for home use the gloves supplied in the package can be considered safe in term of hand exposure during the at-home dyeing procedure, taken into consideration that the exposure time is  limited to 30 minutes. Of course this does not prevent the at-home dye consumer or the person visiting the hairdresser from the risk of sensitization since the major risk is the actual hair dyeing in the individual case.