Contact Dermatitis 2009: 60: 85–90
Printed in Singapore. All rights reserved
#2009 The Authors
Journal compilation#2009 Blackwell Munksgaard
Assessment of the sensitization potential of
persulfate salts used for bleaching hair
MARI´A-JESU´S CRUZ1,2,*, VANESSA DE VOOGHT3,*, XAVIER MUN˜OZ1,2, PETER H. M. HOET3, FERRAN MORELL1,2,
BENOIT NEMERY3AND J. A. J. VANOIRBEEK3
1CIBER de Enfermedades Respiratorias (CIBERES),2Servei de Pneumologia, Hospital Vall d’Hebron, 08035
Barcelona, Spain, and3Unit of Lung Toxicology (Laboratory of Pneumology), K.U. Leuven, B-3000 Leuven, Belgium
Background: Persulfate salts have been associated with both allergic contact dermatitis and bronchial
asthma. Because there is currently no experimental data available on the sensitizing properties of
persulfate salts (ammonium, sodium, and potassium persulfates), we determined their dermal sensi-
tizing capacity, using the murine local lymph node assay (LLNA).
Material and Methods: For three consecutive days, BALB/c mice were dermally treated with ammo-
nium, sodium, or potassium persulfate or with the vehicle alone (dimethyl sulfoxide) on each ear
(2 ? 25 ml). On D6, mice were injected intravenously with [3H]-methyl thymidine. The draining
auricular lymph nodes were removed, and the incorporation of [3H]-methyl thymidine was compared
with that of vehicle-treated control mice. A stimulation index (SI) relative to the vehicle-treated
control value was derived. The sensitizing potency of the chemicals tested was determined by esti-
mating the concentration of chemical required to induce a SI of 3 (EC3).
Results: All three chemicals provoked positive responses in the LLNA, with dose-dependent
increases in proliferation. Maximal SIs recorded were 6.8 ? 1.8, 6.5 ? 1.2, and 5 ? 1.0 at 5% for
ammonium, sodium or potassium persulfate, respectively. The EC3 values were 1.9%, 0.9%, and
2.4% for ammonium, sodium, and potassium persulfates, respectively.
Conclusions: All three persulfate salts need to be considered strong-to-moderate sensitizers according
to the murine LLNA.
Key words: ammonium persulfate; BALB/c mice; local lymph node assay; potassium persulfate;
sodium persulfate. # Blackwell Munksgaard, 2009.
Conflicts of interest: The authors have declared no conflicts.
Accepted for publication 22 July 2008
Persulfate salts are reactive low-molecular-weight
chemical compounds widely used in different
manufacturing processes in the chemical, pharma-
ceutical, metallurgic, textile, photographic, food,
and, particularly cosmetic industries (1). These
salts may be present in hair lightening products
at concentrations up to 60% (2).
Persulfates are capable of causing immunolog-
ical sensitization and subsequent allergic disease,
such as contact dermatitis (3–5) and bronchial
asthma (6–9). Currently, there is a lack of infor-
mation about the skin sensitizing properties of
persulfates. Only the sensitization potential of
ammonium persulfate has been determined in
a commercial setting (10). Ammonium persulfate
was considered a sensitizer according to the
guinea-pig maximization test, an Organization
for Economic Cooperation and Development
(OECD)-approved predictive test using guinea-
pigs for establishing dermal sensitization potential
(guideline no. 406).
The local lymph node assay (LLNA) is a vali-
dated and accepted hazard identification method
that is currently widely used as a predictive test
for the identification of dermal sensitizers. The
*These authors equally contributed to this work.
method is based on the immunological events that
occur during the induction phase of contact sen-
sitization. Contact allergens are identified as
a function of lymphocyte proliferative responses
provoked in draining lymph nodes following
(11, 12), with the degree of lymphocyte prolifera-
tion correlating closely with the skin sensitizing
potency of the chemical (13). One of the main
advantages of this technique is that it permits
accurate measurement of relative potency, thus
allowing an effective risk assessment and risk
management strategy. Furthermore, using the
LLNA, it is possible to distinguish prevalence
(overall frequency of contact dermatitis caused
by a chemical) from potency (determination of
the allergic potential of a chemical, applying a cer-
tain concentration on the skin). This is important
because a relatively high prevalence is not neces-
sarily attributable to a high potency (14).
In a first step to try to identify the mechanisms
involved in the sensitization to persulfate salts, we
have compared, using the murine LLNA, the
sensitization potential of three persulfate salts:
ammonium persulfate, sodium persulfate, and
potassium persulfate. To our knowledge, these
three salts have not been studied using the LLNA.
Materials and Methods
Male BALB/c mice (* 20 g, 6 weeks old) were
obtained from Harlan (Horst, the Netherlands).
The mice were housed in a conventional animal
house with 12:12-hr dark–light cycles. They
received lightly acidified water and pelleted food
(Trouw Nutrition, Ghent, Belgium) ad libitum. All
experimental procedures were approved by the
local Ethical Committee for Animal Experiments.
Ammonium persulfate [(NH4)2S2O8], sodium per-
sulfate (Na2S2O8), and potassium persulfate
(K2S2O8) (Sigma-Aldrich Corporation, St Louis,
MO, USA) were used. Dosing solutions (0.1%,
0.5%, 1%, and 5% w/v) were prepared in
dimethyl sulphoxide (DMSO, vehicle) (Sigma-
Aldrich Corporation). Dosing solutions were pre-
pared freshly for each application.
Local lymph node assay
The LLNA was performed according to a stand-
ard protocol (12). Briefly, the mice (five animals
per test group) received dermal applications of
25 ml of various concentrations of (NH4)2S2O8,
Na2S2O8, or K2S2O8or of the same volume of
vehicle alone on each ear, daily for three consec-
utive days. The area around the exposure site was
monitored for any signs of irritancy. Six days after
the initiation of exposure, the mice were injected
intravenously through the tail vein with (methyl-
3H)-thymidine [3HTdR], 2 Ci/mmol from ICN
Pharmaceuticals (Asse, Belgium) in 250 ml of
(90 mg/kg) and the draining auricular lymph nodes
were excised, pooled for each mouse, and weighed
of lymph node cells (LNC) was prepared, and the
LNC were washed twice.
was measured by b-scintillation counting (Beck-
man LS 5000CE, Irvine, CA, USA) and expressed
as disintegrations per minute (d.p.m.). The SI was
calculated as the ratio of3HTdR incorporation by
lymphocytes from treated animals relative to that
from concurrent vehicle-treated controls.
Data treatment and statistical analysis
The mean value of the d.p.m.s found was calcu-
lated for each experimental group, thus allowing
the derivation of a SI. As threshold level for sig-
nificance a SI value >3 was adopted. An estimate
of the sensitizing potency of the chemicals tested
was determined by calculating an estimated con-
(EC3). The EC3 was derived by linear interpola-
tion of dose–response data as described previously
(15). The EC3 value was calculated by interpolat-
ing between two points on the SI axis, one imme-
SI value of 3. Where the data points lying imme-
diately above and below of 3 have the co-ordinates
(a,b) and (c,d), respectively, then the EC3 value
may be calculated using the following equation:
EC3 ¼ c þ ½ð3 ? dÞ=ðb ? dÞ?ða ? cÞ
The relationship of EC3 value to relative
potency is inverse, hence the lower the EC3 value,
the higher the potency.
Data is shown as means and SD. All data were
analysed using parametric analysis of variance
with a Dunnett’s post hoc test (GRAPHPAD PRISM
4.01 GRAPHPAD Software Inc., San Diego,
CA, USA). A level of P < 0.05 (two tailed) was
considered to be significant.
The skin sensitizing potential of three persulfate
salts was determined using the LLNA, a method
86CRUZ ET AL.
Contact Dermatitis 2009: 60: 85–90
in which the activity is measured as a function of
induced proliferative responses in the auricular
lymph nodes draining the site of topical applica-
tion. In the LLNA, chemicals are defined as bio-
logically relevant dermal sensitizers, when the test
concentrations yield at least a threefold increase in
SI compared with vehicle-treated controls (14).
This is called the EC3. Both the LNW and the
LNC count, measured by3HTdR incorporation
and presented in d.p.m., are presented.
The results of the LLNA for the persulfate salts
are presented in Tables 1 and 2 and Fig. 1.
Exposure to (NH4)2S2O8resulted in a maximal
mean SI of 6.8 ? 1.8 at the highest concentration
tested (5%) (Fig. 1a). Applying 5% solution of
(NH4)2S2O8caused the LNW to increase 2.5 times
(Table 1) and a 6.5-fold increase in total LNC
number (Table 2) was found when compared with
the DMSO control.From thecalculated SIvalues,
the estimated EC3 of (NH4)2S2O8is 1.9%.
After sensitization with Na2S2O8, the maximal
SI of 6.4 ? 1.2 at the highest concentration tested
(5%) was calculated (Fig. 1b), while a threefold
increase in the LNW was measured (Table 1)
and the LNC numbers increased almost 6.5-fold
(Table 2). This resulted in an estimated EC3 of
0.9% for Na2S2O8.
A slightly lower value was obtained after sensi-
tization with K2S2O8, which yielded a maximal
mean SI of 5.0 ? 1.0 for the group exposed to
the highest concentration (5%) (Fig. 1c). The
LNW increased 2.8-fold, while the total LNC
number was five times increased. This resulted in
an estimated EC3 of 2.4% for K2S2O8.
The primary purpose of our study was to inves-
tigate the potential of three persulfate salts
[(NH4)2S2O8, Na2S2O8, or K2S2O8] to cause aller-
gic contact dermatitis. Thisis necessary for further
studying the potential of these salts to induce pul-
monary effects and, moreover, to investigate the
relation between dermal sensitization and res-
piratory responses (16–19). Kimber et al. (20)
proposed a classification to categorize contact
allergens according to potency, based on their
EC3 value. In this approach, four categories are
used. 1, extreme skin sensitizers are defined as
agents with an EC3 value less than 0.1%; 2, strong
sensitizers have an EC3 value between 0.1% and
1%; 3, moderate sensitizers have an EC3 value
between 1% and 10%; and 4, weak sensitizers
have an EC3 value above 10%. In this classifica-
tion, (NH4)2S2O8 (EC3 ¼ 1.9%) and K2S2O8
(EC3 ¼ 2.4%) are classified as moderate sensi-
tizers and Na2S2O8(EC3 ¼ 0.9%) is classified as
a strong sensitizer.
The information on the sensitizing properties of
persulfates is limited. One study describes the
safety assessment of ammonium, potassium, and
sodium persulfate (2). In that study, only ammo-
nium persulfate was tested for its dermal sensitiz-
ing potential, using the Magnusson and Kligman
guinea-pig maximization test (21), which is an
OECD-approved guideline (no. 406, 1992) for
Table 1. Lymph node weights in response to treatment with
vehicle (DMSO), ammonium persulfate [(NH4)2S2O8], sodium
persulfate (Na2S2O8), and potassium persulfate (K2S2O8)a
2.6 ? 0.9
3.2 ? 0.9
3.5 ? 0.8
4.3 ? 0.9*
6.6 ? 1.4**
3.7 ? 1.1
3.9 ? 1.3
4.8 ? 1.6
6.7 ? 0.9**
11.6 ? 1.8**
3.4 ? 1.7
4.1 ? 0.7
4.5 ? 1.2
7.5 ? 2.2**
9.5 ? 2.3**
DMSO, dimethyl sulphoxide; LNW, lymph node weight.
aIn the murine local lymph node assay, mice were dermally
sensitized, on both ears, on D1, D2, and D3. On D6, they were
intravenously injected with 20 mCi [methyl-3H]-thymidine
(3HTdR), and 5 hr later, the auricular lymph nodes were
removed, pooled, and weighed (lymph node weight). The two
lymph nodes of each mouse were pooled. Numbers are means
(?SD) of 4–7 mice per group. *P < 0.05, **P < 0.01 compared
with the DMSO control.
Table 2. Disintegrations per minute (d.p.m.) in the LLNA in response to treatment with vehicle (DMSO), ammonium persulfate
[(NH4)2S2O8], sodium persulfate (Na2S2O8), and potassium persulfate (K2S2O8)a
Concentration (% w/v)(NH4)2S2O8(in DMSO) d.p.m. Na2S2O8(in DMSO) d.p.m.K2S2O8(in DMSO) d.p.m.
1609 ? 681
1424 ? 720
2116 ? 250
2942 ? 475*
10 513 ? 2739**
1556 ? 870
1488 ? 547
2284 ? 357
5596 ? 484**
10 082 ? 1936**
1630 ? 284
1579 ? 240
2130 ? 323
3145 ? 649**
8179 ? 1568**
DMSO, dimethyl sulphoxide; LLNA, local lymph node assay.
aIn the murine LLNA, mice were dermally sensitized, on both ears, on D1, D2, and D3. On D6, they were intravenously injected with
20 mCi [methyl-3H]-thymidine (3HTdR), and 5 hr later, the auricular lymph nodes were removed, pooled, and weighed (lymph node
weight).3HTdR incorporation was measured by b-scintillation counting expressed as d.p.m.. The two lymph nodes of each mouse
were pooled. Numbers are means (?SD) of 4–7 mice per group. *P < 0.05, **P < 0.01 compared with the DMSO control.
Contact Dermatitis 2009: 60: 85–90
SENSITIZATION POTENTIAL OF PERSULFATE SALTS87
testing dermal sensitization. In that study, all
the guinea-pigs reacted to an intradermal admin-
istration of a 0.1% solution in saline, whereas
80% of the animals reacted to epicutaneous ap-
plication of a 1% solution. According to these
results, (NH4)2S2O8was considered a dermal sen-
sitizer for the guinea-pig (2). Mensing et al. (22)
demonstrated in a rabbit model that exposure to
hair bleach containing persulfates increased air-
way hyper-responsiveness and that ammonium
persulfate stimulated the release of nitric oxide
in guinea-pigs (23).
Murine LLNA is an OECD-accepted method
(OECD guideline no. 429, 2002) to identify con-
tact sensitizers. The argument is that skin expos-
ure to a sufficient amount of a relevant chemical
allergen will induce an immune response of the
quantity and quality necessary to cause systemic
sensitization (24). In this present set-up, we
slightly adjusted the murine LLNA. Instead of
using CBA/Ca mice as described in OECD guide-
line 429, we used BALB/c mice. The purpose of
this study was to investigate the dermal sensitizing
potential of persulfate salts. This study will be
elaborated with a second study in which the
potential of these salts to induce respiratory sen-
sitization and their ability to induce asthma are
described. For studies of these pulmonary effects
induced by a chemical, BALB/c mice are the pre-
ferred mouse strain (16–19).
According to the results obtained, (NH4)2S2O8,
K2S2O8, and Na2S2O8, which are known for their
lung and skin sensitization potential in humans
nium and potassium persulfate can be categorized
as moderate sensitizers, while sodium persulfate
should be regarded as a strong sensitizer.
Differences between the persulfate salts have
also been observed previously in several studies.
Parsons et al. (25) reported that both ammonium
and potassium persulfates caused histamine
release from isolated rat peritoneal mast cells
and from guinea-pig skin in vitro and in vivo. His-
tamine release induced by potassium persulfate
was characterized by degranulation of the mast
cells. However, with ammonium persulfate, alter-
ationsin thegranules were observed butno appar-
ent degranulation occurred. In vitro studies with
slices of guinea-pig skin, potassium persulfate but
not ammonium persulfate, appeared to release
Several studies have already been conducted
to relate LLNA EC3 values with skin sensitiza-
tion based on clinical observations. These stud-
ies show a very close correlation between clinical
observations and estimates of potency based on
EC3 values (26, 27). The LLNA results obtained
in the present study are consistent with the well-
known ability of persulfates to cause sensiti-
zation among exposed human populations.
SI ammonium persulfate
EC3 = 1.9
SI sodium persulfate
EC3 = 0.9
SI potassium persulfate
EC3 = 2.4
Fig. 1. Stimulation index (SI) of ammonium (a), sodium (b),
and potassium (c) persulfate in the murine local lymph node
assay. On D1, D2, and D3, mice were dermally sensitized on
both ears. On D6, they were intravenously injected with
20 mCi [methyl-3H]-thymidine (3HTdR), and 5 hr later, the
auricular lymph nodes were removed, pooled, and weighed.
The SI was calculated as the ratio of3HTdR incorporation
by lymphocytes from treated animals relative to that from
concurrent vehicle-treated controls. (NH4)2S2O8, Na2S2O8,
and K2S2O8persulfates were dissolved in dimethyl sulfoxide.
The two lymph nodes of each mouse were pooled. Symbols
represent means (?SD) of 4–7 mice per group. *P < 0.05,
**P < 0.01 compared with the DMSO control.
88CRUZ ET AL.
Contact Dermatitis 2009: 60: 85–90
In humans, persulfates were reported to cause
both delayed-type and immediate skin reactions,
including irritant dermatitis, allergic eczematous
generalized urticaria (1, 5). In addition to skin
diseases, respiratory disorders have been fre-
quently reported among hairdressers and chem-
ical workers exposed to persulfates (6–9).
Persulfate salts are frequently implicated agents
in the origin of occupational asthma that may
represent up to 4% of all occupational asthma
In conclusion, we provided here a systematic
comparative analysis of three persulfate salts of
occupational significance using the LLNA. The
study shows that all three persulfate salts, ammo-
nium, sodium, and potassium, with a potential
for sensitization in humans, were positive in the
according to EC3 values showed a (limited) dis-
tinction between ammonium and potassium per-
sulfates, which are moderate sensitizers, and
sodium persulfate, which is a strong sensitizer.
These data will be of value in developing further
accurate risk assessments.
The project was supported by a grant of the Inter-
universitary Attraction Pole Program, Belgian State,
Belgian Science Policy P6/35, from the ‘Fonds voor
Wetenschappelijk Onderzoek Vlaanderen’ (FWO),
FWO G.0547.08, from the Fundacio ´ Catalana de
Pneumologia, from the Societat Catalana de Pneumo-
logı´a, and from PIS PI080730 J. A. J. V. is a post-
doctoral fellow of the FWO.
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Dr Marı´a-Jesu´s Cruz
Servei de Pneumologia
Hospital Vall d’Hebron
Pg. Vall d’Hebron 119
Tel: þ34 93 4894048
Fax: þ34 93 4894049
90 CRUZ ET AL.
Contact Dermatitis 2009: 60: 85–90