Pilot study: Role of anti-oxidants in the elicitation of contact allergic reaction to p-phenylenediamine (PPD).

Allergic contact dermatitis is a T cell-mediated delayed type hypersensitivity
induced by allergens such as PPD, resulting in a skin reaction. Stimulation
with contact allergens generates ROS (reactive oxygen species) and causes
oxidative degradation of hyaluronic acid. This results in indirect toll-like
receptor activation and eventually amplification of the in*ammatory response
resulting in a rash . The topical application of the antioxidant luteolin
prevented sensitization and elicitation in a contact hypersensitivity (CHS)
model (3). Induction of ROS and their role in the CHS response was identified
by measurement of ROS production in dendritic cells in vitro and in vivo in
murine skin (3,4). Antioxidants and hyaluronidase inhibitors were used to
analyse the role of ROS and HA degradation in vitro and in vivo. Generation and
degradation of hyaluronic acid as a result of ROS formation was studied in the
skin by immunohistology.

Over a decade ago, Kuriyama et al demonstrated that vitamin E ointment at
20-40% suppressed allergic and irritant contact dermatitis in rats (5). This
vitamin E ointment seemed to stabilize the keratinocytes and prevented the
development of erythema.

Studies in mice demonstrated that topical pre-treatment with anti-oxidants
prevents allergic contact dermatitis and even application of anti-oxidants
after the contact with the allergen, prevented the allergic dermatitis (4). An
explanation for this is the blockage of ROS. ROS crucially contributes to
contact sensitizer induced skin inflammation and blocking of ROS production.
This effectively inhibits contact hypersensitivity responses both in the
sensitization as well as in the elicitation phase.

By applying an anti-oxidant cream before the usage of hair dye, the elicitation
reaction might be diminished by the inhibition of ROS and result in no or a
less severe skin reaction to PPD.

A simple self-test with a patch on their forearm may give an indication about
such a contact-allergy, which was investigated in a pilot study conducted in
the UMCG in 2007. A use test of 30 minutes was enough to demonstrate a positive
reaction in 53-60% of the subjects with a ++ reaction in the past and in
93-100% of the patients with a +++ reaction.

An unpublished study in humans demonstrated a reduction of elicitation response
to 0.1%, 0.5%, 1% hair dye and 1% PPD after pretreatment with vitamin C.
However, the procedures used in this study, were not standardized. In addition,
the use condition of 2% PPD was not used in the previous study. Therefore we
would like to study the effect of a vitamin C product (pre)treatment on
elicitation reaction to hair dye product with 2% PPD.

The current study will apply the same protocol as the pilot study from 2007,
but will in addition to the use test for hair dyes, include a pretreatment with
an anti-oxidant solution. Anti-oxidants are expected to prevent the elicitation
reaction due to coupling between the PPD and the anti-oxidant, which makes the
PPD less likely to penetrate the skin.

(1) Thyssen JP, White JM, European Society of Contact Dermatitis.
Epidemiological data on consumer allergy to p-phenylenediamine. Contact
Dermatitis 2008:59:327-343.
(2) McFadden JP, White IR, Frosch PJ, Sosted H, Johansen JD, Menne T. Allergy
to hair dye. BMJ 2007:334:220.
(3) Esser PR, Woelfle U, Jakob T, Schempp CM, Martin SF. Linking ROS production
and HA degradation - A crucial role for the generation of endogenous ligands in
CHS responses to contact sensitizers. Experimental Dermatology 2011:20:177.
(4) Esser PR, Wolfle U, Durr C, von Loewenich FD, Schempp CM, Freudenberg MA,
et al. Contact sensitizers induce skin inflammation via ROS production and
hyaluronic acid degradation. PLoS One 2012:7:e41340.
(5) Kuriyama K, Shimizu T, Horiguchi T, Watabe M, Abe Y. Vitamin E ointment at
high dose levels suppresses contact dermatitis in rats by stabilizing
keratinocytes. Inflamm Res 2002:51:483-489.

Pilot study to investigate the influence of pre-treatment with an anti-oxidant
solution on the elicitation reaction (the occurrence of an allergic skin
reaction after contact with an allergen) in subjects with a proven
sensitization to PPD.

For this study, we aim to include the same subjects as in the pilot study,
given the fact that they have been extensively tested and documented. In total
30 subjects will be included.
A short interview will be held to confirm the inclusion and exclusion criteria.
Since the included subjects already participated in a similar patch-testing
study before, this will only be a short check. Special attention will be paid
to new immunosuppressive medication and pregnancy will be excluded by history.
No clinical tests will be performed to exclude pregnancy or co-morbidity.
The volar side of the forearms will be inspected for active skin disease by
means of physical examination.

Eight different test areas are marked on the volar aspects of the forearms (4
on each arm from 2 by 2 cm). The anti-oxidant cream will be applied to three
test areas and the anti-oxidant free cream will be applied to three other test
areas. Two test areas will not be pre treated. After 10 minutes 0% and 2% PPD
hair dye cream will be applied to 6 out of the 8 test areas for 30 minutes,
resulting in 8 different combinations:
o 30 minutes 0% PPD hair dye cream mixed with hydrogen peroxide
o 30 minutes 2% PPD hair dye cream mixed with hydrogen peroxide
o 10 minutes antioxidant cream followed by 30 minutes 0% PPD hair dye cream
mixed with hydrogen peroxide (in total 40 minutes antioxidant cream)
o 10 minutes antioxidant cream followed by 30 minutes 2% PPD hair dye cream
mixed with hydrogen peroxide (in total 40 minutes antioxidant cream)
o 10 minutes antioxidant-free cream followed by 30 minutes 0% PPD hair dye
cream mixed with hydrogen peroxide (in total 40 minutes antioxidant-free cream)
o 10 minutes antioxidant-free cream followed by 30 minutes 2% PPD hair dye
cream mixed with hydrogen peroxide (in total 40 minutes antioxidant-free cream)
o 40 minutes antioxidant cream
o 40 minutes antioxidant-free cream

The hair dye is a 1:1 mixture of 4 % PPD, 3,6% 2-methylresorcinol and 1,9%
2-methyl-5-hydroxyethylaminophenol in a basic cream with a 6% solution of water
peroxide. The total PPD concentration is 2%, which is comparable to normal use
conditions and the conditions used in the pilot study of 2008. The 0% PPD hair
dye cream is a negative control, which is the same mixture as the 2% PPD cream,
only without PPD, and with 2-methylresorcinol and
2-methyl-5-hydroxyethylaminophenol. 2-Methylresorcinol and
2-methyl-5-hydroxyethylaminophenol are added as couplers to the hair dye cream
and selected because of their low-sensitization abilities. The basic cream is
Koleston Perfect Chassis without fragrances. The ingredients of this cream are
used in various cosmetics (Lanette O, Dusoran MD, Na-laurylethersulfaat,
Na-cocoyl isethionate, EDTA, Na-sulfite, Ascorbinezuur, Ammonia, Na-hydroxide,
Water).

Forty minutes after the first application, all test areas are rinsed off and
washed with water and shampoo (Andrélon, Iedere dag shampoo, Unilever).
Fifteen minutes after the rinsing (55 minutes after application) the first
reading of the test areas is performed according to the guidelines of the
International Contact Dermatitis Research Group. Recordings of clinical
manifestations will be made. All the tested skin areas will be photographed.
During the study subjects are allowed to continue their daily activities, with
the advice to minimize exposure to sunlight of the tested skin areas until
after visit 3 and extensively rub the test areas. They are allowed to shower,
swim or sport if this is part of their normal routine.
Total duration: 60 minutes

Visit 2, day 2, 48 hours after application
After 48 hours the eight tested skin areas will be inspected again for possible
reactions according to the criteria of the International Contact Dermatitis
Research Group. Recordings of clinical manifestations will be made. All the
test areas will be photographed.
Total duration: 15 minutes.

Visit 3, day 3, 72 hours after application
On day 3 the tested skin areas will be inspected once more for possible
reactions according to the criteria of the International Contact Dermatitis
Research Group. Recordings of clinical manifestations will be made. All the
test areas will be photographed.

On the day of the patch-test application the time burden will be 60 minutes at
the most, in addition to travel time. The actual application of the patch will
cause minimal discomfort. The time burden for reading the patch test reaction
will be less than 15 minutes on day 2 and day 3. There will be a travel time
burden, but the participants are offered the opportunity to perform the
readings at a place of their preference (at their home or at work for example).

Participants might develop a skin reaction due to the PPD. This can exist of
redness, possibly with some itching, vesicles and papules, at the site of the
patch-test with the PPD containing solution. This skin reaction is transient,
and will disappear within a few days. Participants have the option to receive a
one-time application of a corticosteroid-containing cream to enhance the
remission of the reaction. Systemic effects from resorption of PPD are
irrelevant at such a short exposure time and small application area.

All the products used in this study are cosmetics according to the EU
definition: any substance or preparation intended to be placed in contact with
the various external parts of the human body (epidermis, hair system, nails,
lips and external genital organs) or with the teeth and the mucous membranes of
the oral cavity with a view exclusively or mainly to cleaning them, perfuming
them, changing their appearance and/or correcting body odours and/or protecting
them or keeping them in good condition.
The cosmetics are used well within the normal range of use.

Participants will receive gift cards with a value of ¤100,- and reimbursement
of travel expenses.