Identification and Quantification of para

STUDY
Identification and Quantification of
para-Phenylenediamine in a Temporary
Black Henna Tattoo
Ronald R. Brancaccio, Lance H. Brown, Young Tae Chang, Joshua P. Fogelman, Erick A. Mafong, and
David E. Cohen
Background: Temporary black henna tattoos are very popular as body adornment. Although contact allergy to natural henna is
unusual, the inclusion of hair dye, p-phenylenediamine (PPD), increases the risk of contact sensitization.
Objective: This study was performed to identify the presence and concentration of PPD in a black henna tattoo mixture to which
our patient developed contact allergy.
Methods: The presence of PPD in a black henna tattoo mixture, various samples of commercially available henna powders, and
several hair dye products was qualitatively and quantitatively detected using high performance liquid chromatography (HPLC).
Results: This study demonstrated that PPD was present in the black henna tattoo mixture at a concentration of 15.7%, which is
significantly higher than commercial hair dye preparations.
Conclusion: The presence of PPD in black henna tattoo mixtures in high concentration poses a health hazard and a risk of allergic
contact sensitization with potential long-term consequences.
Copyright 2002, Elsevier Science (USA). All rights reserved.
T
HE ART OF BODY ADORNMENT by body piercing
and tattooing has become increasingly popular in
our modern culture. Temporary henna tattoos are readily
available worldwide, last several weeks on the skin, and
offer a self-limited, convenient alternative to a permanent
tattoo. Since ancient times, henna has been used to paint
the skin and dye hair. Although delayed1-3 and immediate4,5 hypersensitivity to henna has been reported, it is
considered a rare allergen.
The addition of p-phenylenediamine (PPD), which is
widely recognized as a sensitizer, increases the risk of
allergic contact dermatitis from these henna tattoo mixtures and a number of cases have been reported.6-11 We
identified the presence of PPD both qualitatively and quantitatively in a black henna tattoo mixture by high-performance liquid chromatography (HPLC).
Case Report
A 37-year-old woman painted 2 black henna tattoos on her
left upper arm and lower back. Within 24 to 48 hours, a
pruritic dermatitis developed within the designs of both
tattoos. Oral prednisone and topical clobetasol proprionate
cream were given, and resolution occurred in 2 weeks
(Fig 1).
Ten years before presentation, the patient developed an
From the Ronald O. Perelman Department of Dermatology, New York University
Medical Center and the Department of Chemistry, New York University, New York, NY.
Address reprint requests to Ronald R. Brancaccio, MD, 67 Perry St, New York, NY
10014.
Copyright 2002, Elsevier Science (USA). All rights reserved.
1046-199X/02/1301-0004$35.00/0
doi:10.1053/ajcd.2002.30466
allergic reaction in her scalp following the use of a hair dye,
which also required oral prednisone for treatment. Previously, she had worked as a hairdresser, but did not recall
any problems with exposure to hair dyes. She denies any
recent occupational exposure to PPD and currently works
as a management consultant.
Patch testing was performed on the inner aspects of her
arms using Finn Chambers on Scanpor tape (Allerderm,
Laboratories, Petaluma, CA). The materials tested included the black henna tattoo mixture, reconstituted with
tap water to a paste consistency as directed by the inserted
package instructions; PPD 1% pet. (Chemotechnique, Chemotechnique Diagnostics, AB, Malmö, Sweden); lawsone
1.0% in petrolatum; and 3 different preparations of commercially available henna powders, each at a 1.0% and
10.0% aq. concentration.
Within 7 hours, the patch test sites of PPD and the black
henna tattoo mixture became so severely pruritic that the
patient removed them from her left arm. At 24 hours after
application, strongly positive 3⫹ (erythema, edema, and
vesicles) reactions were noted at both sites (Fig 2). These
reactions persisted and remained strongly positive by the
second reading at 1 week. The patch test results to lawsone
and the henna mixtures on the right arm were negative at
48 hours and at 1 week. No reactions were noted in 10
control patients patch tested with lawsone and the 3 henna
samples.
Materials and Methods of HPLC
A stock solution of PPD (Aldrich Chemical Co, Milwaukee,
WI) was made in dimethylsulfoxide (DMSO), 10 mg/mL
(Fisher Scientific, Pittsburgh, PA). Dilutions were made in
acetonitrile (Fisher Scientific, Pittsburgh, PA) to give 10, 5,
American Journal of Contact Dermatitis, Vol 13, No 1 (March), 2002: pp 15-18
15
16
Brancaccio et al
Discussion
Figure 1. Allergic contact dermatitis to black henna tattoo.
Henna tattooing is an ancient skin decorating custom of
middle eastern and south Asian cultures. Henna is a natural substance derived from leaves and flowers of the plant
Lawsonia inermis. Lawsone, a naphtoquinone, is the active
coloring ingredient and considered the allergen in henna.1
The dry leaves of the plant are ground into a fine powder
and mixed with oil or water and applied to the skin for 2 to
6 hours. Substances such as lemon, clove oil, or PPD are
added to henna to decrease the application time from
hours to minutes.2 These painless tattoos last for about 3
weeks. Traditional henna tattoos stain the skin a redbrown color.
PPD is added to henna tattoos not only to accelerate the
dyeing process, but also to strengthen and darken the
and 1 ␮g/mL. HPLC was performed using the HP 1100
HPLC machine (Hewlett Packard, Palo Alto, CA) equipped
with a diode array ultraviolet (UV) detector and electrospray mass-spectrometry detector. The HPLC mass spectrometry condition was determined (0.1% MeCN, 0.1%
AcOH in water, 0.4 mL/min, Luna 50⫻2.0 mm, 5␮ C18).
Retention time of PPD was about 0.26 minutes in this
condition. A standard curve was made using 10, 5, and 1
␮g/mL of PPD. Real samples that included black henna
tattoo mix, 3 different henna powders, Bigen oriental black
#59 hair color, Clairol Beautiful Collection permanent
hair color, Clairol Loving Care semipermanent hair color,
and Colora henna cream hair color were dissolved in
DMSO to give 10 mg/mL solution. These were then diluted
in acetonitrile to give 1000, 100, and 10 ␮g/mL. A 100- or
1000-␮g/mL solution was used to determine the PPD
amount. The raw integration value at 235 nm was converted into percentage weight of PPD in the sample using
a standard curve. All of the positive peaks were checked by
mass spectrometry and UV spectrum to confirm the identity (i.e., PPD). Samples positive for PPD underwent repeat
HPLC testing and a mean and standard deviation were
calculated.
Results
The results of HPLC are summarized in Table 1. The black
henna tattoo mixture contained an average of 15.7% of
PPD by weight, whereas Bigen #59 Oriental Black (Higashi, Nagoya, Japan) hair color contained an average of
12.3% and Beautiful Collection permanent hair color
(Clairol, Stamford, CT) 1.7%. There was no detection
(UND) of PPD at 1000 ␮g/mL in the 3 commercially
purchased (in New York and California) henna samples,
indicating a PPD content less than 0.1%. Similarly, PPD
was not identified in Loving Care semipermanent hair
color (Clairol, Stamford, CT) and Colora Henna Cream
(Colora, NY).
Figure 2. Positive patch test results to PPD (1% pet.) and black
henna tattoo mix (as is) at 24 hours.
p-Phenylenediamine in a Henna Tattoo
17
Table 1. HPLC
Black Henna Tattoo Mix
Bigen #59
Beautiful
Henna Powder 1
Henna Powder 2
Henna Powder 3
Loving Care
Colora Henna Cream
Mean % Weight of PPD
Standard Deviation
No. of Experiments
15.7
12.3
1.7
UND
UND
UND
UND
UND
0.6
0.7
0.1
UND
UND
UND
UND
UND
4
3
3
1
1
1
1
1
Abbreviations: UND, PPD undetectable at 1,000 ␮g/mL (PPD content ⬍ 0.1percent).
color, and enhance the design pattern of the tattoo. These
tattoos stain the skin black and have the appearance of a
real tattoo. Allergy to PPD may occur from prior exposure,
as probably occurred in our patient, or may develop during
the tattooing process. An increasing number of reports of
contact allergy from temporary black henna tattoos have
appeared in the dermatologic literature, and most of these
have implicated PPD as the causative allergen by inference, in that the patients reacted to PPD on patch testing.6-11
The use of HPLC allowed the determination, both
qualitatively and quantitatively, of the presence of PPD in
a black henna tattoo mix in this case. Although the presence of PPD in black henna tattoos has been suspected
previously, this is the first report to identify and determine
the quantity in percent by weight of PPD. The concentration of PPD present, 15.7%, is considerably higher than the
recommended concentration of up to 5.0% of PPD in
hair-coloring products.12 The concentration of PPD in this
black henna tattoo mixture is almost 10 times greater than
this commonly available Beautiful by Clairol black hair
dye. Bigen #59 Oriental black hair color has a high concentration of PPD (12.3%), and may be used by tattoo
artists for mixing with henna. The source of our patient’s
tattoo mixture was a tattoo parlor in Manhattan, but its
origin is unknown. Because black henna tattoo mixtures
are extemporaneouly prepared by the artist with a variety
of materials and sources, the actual concentration of PPD
may vary greatly.
Because of its allergenicity, several countries, including
France, Germany, and Sweden, have banned the use of
PPD as a hair dye. The European Union maximum allowable PPD concentration in hair care products is 6.0%, and
the use of PPD and its derivatives for dying eyelashes,
eyebrows, or the skin is prohibited. In the United States, its
use as a hair dye is not restricted, provided that the product
is labeled to warn of potential dermatitis and gives instructions for patch testing according to section 601(a) of the
Federal Food Drug and Cosmetic Act.13 The caution statement reads as follows: “Caution: This product contains
ingredients which may cause skin irritation on certain
individuals and a preliminary test according to accompa-
nying directions should be made. This product must not be
used for dyeing the eyelashes or eyebrows; to do so may
cause blindness.”
According the Food and Drug Administration (FDA),
the only legal use of PPD in cosmetics is as a hair dye. It is
not approved for direct application to the skin.13 An import
alert is now in effect for foreign-made temporary tattoos
because they do not have the required ingredient declaration on the label or they contain colors not permitted for
use on the skin. Henna itself also is approved only as a hair
dye and not as a skin paint. The Scientific Committee for
Cosmetic Products and Non-Food Products Intended for
Consumer Use (SCCNFP) also has stated that PPD and
similar chemicals should not be used in temporary tattoos.14
Both PPD-sensitized individuals as well as those not
previously sensitized are susceptible to contact allergy from
these tattoo mixtures. The high concentration of PPD in
this black henna tattoo (15.7%) may increase the risk of
sensitization. At only a 10% concentration, PPD sensitized
all 24 subjects tested in a human maximization study.15
Other predictive assays have also confirmed its sensitizing
potential.16 Prolonged contact with the skin also increases
the risk of active sensitization, as these tattoo mixes do not
contain an oxidizing agent as do hair dyes.
Temporary as they are, black henna tattoos may have
permanent sequalae, including lifelong allergy to hair dye
and related allergens, scarring of the tattoo site,17 and
postinflammatory pigment alteration.8 This sensitization
can affect occupational decisions and limit career choices.
The most severe reactions to this combination of PPD and
henna in tattoos have been reported in Sudanese women.18
This syndrome begins with angioneurotic edema and
progresses to acute renal failure and death caused by renal
tubular necrosis.
Although prohibited for sale to consumers by the
FDA, black henna tattoo mixtures also should be banned
for use by tattoo artists. The traditional method of
henna tattooing using only the natural plant powders
without additives would be much safer and limit the risk
of contact allergy.
18
Brancaccio et al
References
1. Gupta BN, Mathur AK, Agarwal C, et al: Contact sensitivity to henna.
Contact Dermatitis 15:303-304, 1986
2. Nigram PK, Saxena AK: Allergenic contact dermatitis from henna. Contact Dermatitis. 18: 55-56, 1988
3. Garcia Dritz JC, Terron M, Bellido J: Contact allergy to henna. Int Arch
Allergy Immunol 114:298-299, 1997
4. Cronin E: Immediate-type hypersensitivity to henna. Contact dermatitis
5:198-199, 1979
5. Majoie IN, Bruynzeel DP: Occupational immediate-type hypersensitivity
to henna in a hairdresser. Am J Contact Dermat 7:38-40, 1996
6. Le Coz CJ, Lefebvre C, Keller F, et al: Allergic contact dermatitis caused
by skin painting (pseudotattooing) with black henna, a mixture of henna
and p-phenylenediamine and its derivatives. Arch Dermatol 136:15151517, 2000
7. Sidbury R, Storrs FJ: Pruritic eruption at the site of a temporary tattoo.
Am J Contact Dermat 11:182-183, 2000
8. Tosti A, Pazzaglia M, Corazza M, et al: Allergic contact dermatitis caused
by mehindi. Contact Dermatitis 42:356, 2000
9. Mohamed M, Nixon R: Severe allergic contact dermatitis induced by
para-phenylenediamine in paint-on temporary ‘tattoos.’ Australas J Dermatol 41:168-171, 2000
10. Tosti A, Pazzaglia M, Bertazzoni M: Contact allergy from temporary
tattoos. Arch Dermatol 136:1061-1062, 2000 (letter)
11. Nixon R, Orchard D: Positive para-phenylenediamine (PPD) reactions
following paint-on tattoos. Australas J Dermatol 40:120, 1999
12. 1999 CIR Compendium: Cosmetic Ingredient Review, Washington, DC.
Final report on the safety assessment of p-phenylenediamine. J Am College
Toxicol 4: 203-214, 1985
13. US Food and Drug Administration: Office of Cosmetics and Colors Fact
Sheet. Available at: http://www.cfan.fda.gov/⬃dms/cos-tatt.html. Accessed:
June 8, 2001
14. SCCNFP Position Statement. [March 13, 2001.] Available at: http://
europa.eu.int/comm/food/fs/sc/sccp/out140-en.html. Accessed: June 8,
2001.
15. Kligman A: The identification of contact allergens by human assay.
J Invest Dermatol 47:393-402, 1966
16. Basketter DA, Gerberick GF, Kimber I, et al: The local lymph node assay.
Available alternative to currently accepted skin sensitization tests. Food
Chem Toxicol 34:985-997, 1996
17. Lewin PK: Temporary henna tattoo with permanent scarification. JAMC
160:310, 1999 (letter)
18. Nater JP, DeGroot AC, Liem DH: Unwanted Effects of Cosmetics
and Drugs Used in Dermatology (ed2). Amsterdam, Elsevier Science,
1985