Received: 13 March 2020 | Revised: 14 May 2020 | Accepted: 14 May 2020

DOI: 10.1111/jocd.13502

ORIGINAL CONTRIBUTION

Skin irritation potential of cosmetic preservatives: An

exposure-relevant study

Xue Ma MSc1,2 | Huan Wang MSc1,2 | Yanqing Song MSc1,2 | Yao Pan PhD1,2

1
Department of Cosmetics, School of
Science, Beijing Technology and Business
University, Beijing, China
2
Beijing Key Laboratory of Plant Research
and Development, Beijing, China
Correspondence
Yao Pan, Department of Cosmetics, School
of Science, Beijing Technology and Business
University, 11 Fu Cheng Road, Hai Dian
District, Beijing, 10048, China.
Email: panyao@btbu.edu.cn
Funding information
Scientific Research Project of Beijing
Educational Committee, Grant/Award
Number: KM201810011008; National
Natural Science Foundation of China, Grant/
Award Number: 81903361
Abstract
Background: Preservatives represent one of the main causes of skin irritation and
contact allergies.
Aims: To comprehensively evaluate the skin irritation potential of phenoxyethanol,
methylparaben, propylparaben, imidazolidinyl urea, and DMDM hydantoin under
regulatory acceptable concentrations.
Methods: A patch test and repeated open application test (ROAT) were applied to
evaluate skin irritation in vivo. In vitro alternative methods consisting of the keratino-
cyte cytotoxicity assay, red blood cell (RBC) test, and hen's egg test-chorioallantoic
membrane (HET-CAM) were performed to elucidate the mechanism of preservative-
induced irritation responses.
Results: The patch test showed that all test substances showed a weak erythema re-
sponse. Propylparaben had the highest occlusive irritancy potential in the patch test,
owing to damage to the cell membrane. The two formaldehyde releasers showed
noticeable skin irritation potential in the ROAT through their cytotoxicity to keratino-
cytes, while a visible response was observed after applying phenoxyethanol and the
two parabens. No filtration was noticed in the in vivo tests, which might be attributed
to the failure of subcutaneous vessel alteration by the preservatives.
Conclusions: Commonly used cosmetic preservatives have minor skin irritation po-
tential with mild erythema reaction under practical use, especially formaldehyde re-
leasers and propylparaben.

KEYWORDS

alternative methods, preservatives, regulatory acceptable concentration, skin irritation

1 | I NTRO D U C TI O N
Cosmetic products comprise a variety of nutrient-rich ingredients
that favor microbial growth, and the microbial contamination of
cosmetics is a health risk for consumers. Preservatives are an es-
sential component to inhibit the development of microorganisms
1
and to prolong the shelf life and usage time of cosmetic products.
Unfortunately, preservatives have been reported to cause skin irrita-
tion and be a common source of allergies in cosmetic and household
products. 2-4 Based on the frequency of use and the prevalence of
skin side effects, the prominent preservatives in cosmetics comprise
phenoxyethanol, parabens, formaldehyde releasers, isothiazoli-
nones, and organic acids.5,6 Their health effects are not only due to
the induction of skin irritation but also to their multiple sources of
exposure resulting in both occupational and nonoccupational con-
tact dermatitis.7,8 Considering the widespread use of preservatives in
daily life, it is a great challenge to evaluate the skin irritation potential
of these preservatives and elucidate their underlying mechanisms.

J Cosmet Dermatol. 2020;00:1–9. wileyonlinelibrary.com/journal/jocd© 2020 Wiley Periodicals, LLC | 1

2 |
Patch testing is a routine method to ensure adequate safety and
tolerability of cosmetics on human skin. However, many patch test
studies have exposed preservatives to subjects at concentrations
much higher than those normally used in cosmetic product formu-
las.7,9 Furthermore, published positive rates of patch tests were usu-
ally obtained from patients suffering from contact dermatitis.10,11 It
is of great importance to determine the potential irritative response
of skin to preservatives at regulatory, limited concentrations in
healthy people without a prior history of cosmetic-induced contact
dermatitis.
With increasing political and ethical demand to replace animal
experiments, many countries have set regulatory requirements
to evaluate the safety of cosmetic ingredients using nonanimal
test methods, especially in Europe.12 The European Centre for the
Validation of Alternative Methods (ECVAM) and Organization for
Economic Co-operation and Development (OECD) have developed
and adopted many alternative methods to assess the irritation po-
tential of cosmetic ingredients based on cellular responses and mo-
lecular pathways leading to adverse endpoints.13 These assays are
rapid, inexpensive, and high-throughput compared to animal mod-
els and can be used to investigate the toxicological mechanism of
14
chemicals.
In this study, we used the patch test and repeated open applica-
tion test (ROAT), which are two regulatory methods used to evalu-
ate adverse skin effects of cosmetics in China, to examine the skin
irritation potential of five commonly used cosmetic preservatives in
the general population, including phenoxyethanol, methylparaben,
propylparaben, imidazolidinyl urea, and DMDM hydantoin.15 Several
in vitro assays for irritancy were applied to supplement and explore
the possible mechanisms resulting in the in vivo findings. The results
suggested that incorporating in vivo and in vitro nonanimal methods
might be a practical screening platform for assessing and comparing
the cutaneous application safety of cosmetic ingredients.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Chemicals
Phenoxyethanol (99.5%) was obtained from Dow. DMDM hy-
dantoin (97%) was obtained from Macklin Biochemical Co., Ltd.
Methylparaben (99%), olive oil, and petrolatum were obtained from
Aladdin Industrial Corporation. Propylparaben (≥95%) was obtained
from Ark Pharm. Imidazolidinyl urea (98%) and sodium dodecyl sul-
fate (SDS, ≥99%) were obtained from Sigma-Aldrich.
2.2 | Patch test
A total of 32 healthy volunteers (3 males and 29 females) with an av-
erage age of 23.2 ± 1.2 years (range 21-25 years) participated in this
study. All participants were Chinese Hans. The study was conducted
according to the principles of the Declaration of Helsinki. The study
MA et al.
TA B L E 1 Results of human patch test
Total Irritation
Substance (concentration) subjects scorea
Phenoxyethanol (1.0% pet.) 32 0.06 ± 0.04
Methylparaben (0.4% pet.) 32 0.06 ± 0.04
Propylparaben (0.4% pet.) 32 0.13 ± 0.06
Imidazolidinyl urea (0.6% pet.) 32 0.03 ± 0.03
DMDM hydantoin (0.6% pet.) 32 0.06 ± 0.04
Vehicle control (petrolatum) 32 0.00 ± 0.00
Blank control 32 0.00 ± 0.00
a
The values represent the mean ± standard error of irritation score.
was approved by the Ethics Committee of Intertek China on October
23, 2019. Written informed consent to participate in the study was
obtained from each volunteer. The following were exclusion crite-
ria: pregnancy or lactation, any skin disorders or allergies, receipt
of anti-immunologic therapy within 3 months, use of steroid drugs
within 1 month, and lesions on the measurement site. Test proce-
dures and readings were conducted in accordance with Safety and
Technical Standards for Cosmetics (2015 edition).15 Test substances
(25 μL) dissolved in petrolatum were applied in IQ test chambers
(Beijing Baiyi Yida Science and Technology, Beijing, China), and the
test concentrations of each substance are listed in Table 1. The patch
testers were attached to the forearms of subjects for 24 hours. Skin
reactions were evaluated at 30 minutes and 24 hours after patch
tester removal. The reading criteria for skin reactions were as fol-
lows: grade 0 (−), negative reaction; grade 1 (±), doubtful reaction,
with faint erythema only; grade 2 (+), weak positive reaction, with
erythema, infiltration, and possibly papules; grade 3 (++), strong pos-
itive reaction, with erythema, infiltration, papules, and/or vesicles;
and grade 4 (+++), extreme positive reaction, with intense erythema,
infiltration, and coalescing vesicles.
2.3 | Repeated open application test
A total of 30 healthy volunteers (6 males and 24 females) with an av-
erage age of 23.4 ± 1.3 years (range 20-27 years) participated in this
study. All participants were Chinese Hans. The study was conducted
according to the principles of the Declaration of Helsinki. The study
was approved by the Ethics Committee of Intertek China on October
23, 2019. Written informed consent to participate in the study was
obtained from each volunteer. The exclusion criteria were the same
as those listed for the patch test. Test procedures and readings were
conducted in accordance with the European Society of Contact
Dermatitis guidelines for diagnostic patch testing.16 Subjects were
exposed twice daily for 7 days to the test substances, as well as
vehicle controls (water and olive oil). The test concentrations used
for each substance are shown in Table 2. Subjects were thoroughly
instructed on how to apply 50 μL to each 3 × 3 cm2 color-signed
test area on the forearms. Test substance solutions were distrib-
uted evenly within the test area and allowed to dry by evaporation.
MA et al.
TA B L E 2 Results of repeated open application test
Total Total irritation
Substance (concentration) subjects scorea
Phenoxyethanol (1.0% water) 30 0.00 ± 0.00
Methylparaben (0.4% olive oil) 30 0.00 ± 0.00
Propylparaben (0.4% olive oil) 30 0.00 ± 0.00
Imidazolidinyl urea (0.6% water) 30 0.33 ± 0.17*
DMDM hydantoin (0.6% water) 30 0.13 ± 0.09
Vehicle control (olive oil) 30 0.00 ± 0.00
Vehicle control (water) 30 0.00 ± 0.00
Blank control 30 0.00 ± 0.00
a
The values represent the mean ± standard error of total irritation
score.
*P < .05 compared with phenoxyethanol, methylparaben, and
propylparaben.
Subjects could take showers during the ROAT, but were not allowed
to wash the test areas directly. Skin reactions on the test areas were
evaluated each day before the second application. The ROAT skin
reaction of the application area was graded as follows: 0 = no vis-
ible reaction; 1 = weak and spotty erythema (<25% application area);
2 = medium and homogeneous erythema, papules, or both (25%-49%
application area); 3 = strong erythema, papules, and slight vesicles
(50%-89% application area); and 4 = strong erythema, homogeneous
infiltration of papules, and confluent vesicles (90%-100% application
area). The total irritation score of each test compound was deter-
mined for each subject by summing the scores of each day. A mean
total score was then determined for all the subjects.
2.4 | Keratinocyte cytotoxicity assay
Normal human epidermal keratinocytes (NHEKs) obtained from
human foreskin samples were purchased from Guangdong BioCell
Biotechnology Co. Ltd., and were maintained in KcGrowth culture
medium (Guangdong BioCell Biotechnology) at 37°C in a humidified
atmosphere containing 5% CO2. Cells were subcultured with 0.25%
trypsin (Gibco) when they reached 60% confluence. Cytotoxicity
was assessed using a 3-[4,5]dimethylthiazol-2,5dephenyltetrazolium
bromide (MTT) assay based on the reduction in the soluble yellow
MTT tetrazolium salt to its blue insoluble MTT formazan product by
mitochondrial succinic dehydrogenase. Briefly, NHEKs were cultured
for 24 hours in 96-well plates at 1 × 10 4 cells/well. After removing
the culture medium, cells were treated with test substance dosing
medium at concentrations ranging from 7.8 to 1000 μg/mL (methyl-
paraben, propylparaben, imidazolidinyl urea, and DMDM hydantoin)
or from 78.1 to 10 000 μg/ml (phenoxyethanol) for 24 hours. MTT
(5 mg/mL) was then added to each well, and the cells were further
incubated for 4 hours at 37°C. After incubation, a lysis solution (10%
SDS, 5% isobutanol, and 12 mM HCl in H2O) was added to each
well to dissolve the formazan product. Absorbance was determined
at 570 nm using a Tecan Infinite M200 Pro multimode microplate
| 3
reader (Tecan Trading AG, Switzerland). The cell viability data are ex-
pressed as a percentage of the vehicle control. Each experiment was
repeated 3 times, and each treatment group had 6 replicate wells per
experiment. The IC50 (concentration required to inhibit 50% of cell
viability) for each test substance was calculated by nonlinear regres-
sion analysis of data using GraphPad Prism software.
2.5 | Red blood cell test
The red blood cell (RBC) test was performed according to ECVAM
DB-ALM Protocol No.37 using rabbit erythrocytes to detect cell
membrane damage (hemolysis).17 Blood samples were withdrawn
from the marginal vein of one healthy New Zealand white rabbit
purchased from Beijing Xinglong Experimental Animals Co. Ltd.
(Experimental Animal Production License No. SCXK (Beijing) 2016-
0003, China). An RBC suspension was prepared by washing with PBS
(pH 7.4) three times. Each test substance was tested in triplicate. Test
samples were dissolved in PBS, and 25 μL RBC suspension (8 × 109
cells/mL) was mixed with 975 μL test sample solution. The mixture
was agitated at 150 r/min for 10 minutes at room temperature and
centrifuged at 10 000 g for 1 minute. Deionized water served as the
positive control, PBS as the negative control and SDS solutions rang-
ing in concentrations from 0 to 80 μg/mL as the hemolysis reference.
The absorbance of the supernatant at 530 nm was measured, and
the half-maximal effective concentration (H50) for hemolysis was
determined from the dose-response curve to evaluate acute eye ir-
ritancy potential.
2.6 | Hen's egg test-chorioallantoic membrane
The hen's egg test-chorioallantoic membrane (HET-CAM) test was
conducted according to ECVAM DB-ALM Protocol No. 96.18 Briefly,
fertile white Leghorn eggs were incubated with rotation (at least
three times a day) at 37°C and 65% RH in an egg incubator (BSS160)
for 9 days. On day 9 of incubation, viable eggs with normally de-
veloped embryonic vascular systems were selected for treatment.
The eggshell was opened on the side of the air chamber, and the
inner membrane in contact with the CAM was moistened with 2 mL
of 0.9% NaCl solution (saline). A reaction-time method was adopted
in this study with each substance tested on six prepared eggs.
Approximately 150 μl aliquots of test substances dissolved either
in saline or olive oil were applied onto the CAM. Saline and olive oil
were used as negative controls, and 0.1 M NaOH was used as a posi-
tive control. During a period of 300 seconds (5 minutes), the first ap-
pearance of the three endpoints (C-coagulation, H-hemorrhage and
L-lysis of blood vessels) under a stereomicroscope (S8 APO, Leica)
was recorded in seconds. The irritation score (IS) was calculated ac-
cording to previously described methods.18 An IS between 0 and 0.9
predicts no irritation, that between 1 and 4.9 predicts weak or slight
irritation, a score from 5 to 8.9 predicts moderate irritation, and that
from 9 to 21 predicts strong or severe irritation.
4 |
F I G U R E 1 Dose-response curves of cytotoxicity induced by five preservatives in NHEK cells with MTT assay. NHEK cells were treated
with 7.8-1000 μg/mL concentrations of methylparaben, propylparaben, imidazolidinyl urea, and DMDM hydantoin and 78.1 - 10 000 μg/mL
concentrations of phenoxyethanol for 24 hours. Data were shown as mean ± standard deviation (n = 3)
2.7 | Statistical analysis
Statistical analysis was performed using GraphPad Prism soft-
ware (GraphPad). Multiple comparisons of the ISs of each pre-
servative were analyzed with one-way ANOVA followed by the
Bonferroni test. Statistics with a P-value <.05 were considered
significant.
3 | R E S U LT S
3.1 | Patch test: evaluation of primary skin irritation
potential of preservatives
In the 24-hours occlusive patch test, the mean skin reaction
score of the tested preservatives is shown in Table 1. Petrolatum
was used as the vehicle due to the low irritation rate and high
solubility of all the test substances. All the subjects had no ad-
verse skin reactions to the petrolatum and the test chamber,
indicating that the vehicle and the patch material were both
safe to the skin. To simulate the normal use exposure level, we
applied preservatives at the regulatory maximum acceptable
concentrations according to Chinese legislation that authorizes
preservatives as restricted substances in cosmetics.15 Only mild
erythema of the patch area was observed after exposure to the
five preservatives, and there was no significant difference in ISs
between them. Propylparaben had the highest mean IS of 0.13.
The scores of phenoxyethanol, methylparaben, and DMDM
MA et al.
hydantoin were only half of the highest score, and imidazolidi-
nyl urea was a quarter of the highest. This result suggested that
the primary skin irritation response caused by propylparaben
in cosmetics was more common than that of the other four
preservatives.
3.2 | ROAT: evaluation of cumulative skin irritation
potential of preservatives
To further clarify the relevance of patch test reactions induced by
preservatives in the daily use of cosmetics, ROAT was conducted
on 30 healthy subjects, and the total IS results are presented in
Table 2. As petrolatum is sticky and hard to evenly spread on the
skin, we used water or olive oil to dissolve the test substances based
on their solubility. None of the participants reacted positively to the
two vehicle controls during the test period, indicating that water and
olive oil were suitable vehicles in the ROAT. We observed spotty
erythema over the application area in response to imidazolidinyl
urea and DMDM hydantoin, with mean total ISs of 0.33 and 0.13,
respectively. However, the subjects failed to respond to phenox-
yethanol, methylparaben, and propylparaben in the ROAT, which
was different from the patch test. In addition, the total IS of imida-
zolidinyl urea was significantly higher than that of phenoxyethanol,
methylparaben, and propylparaben. After repeated exposure to the
preservatives for 7 days, the irritating reactivity to the formaldehyde
releasers imidazolidinyl urea and DMDM hydantoin was prominent
among the participants.
MA et al. | 5

3.3 | Keratinocyte cytotoxicity assay: the
impact of the preservatives on skin cell viability
According to the pathogenesis of skin irritation, NHEK cells are suit-
able for testing the irritation potential of chemicals intended for der-
matological use in vitro. The cell viability of NHEK cells decreased in
a dose-dependent manner following exposure to the preservatives
at the tested concentrations for 24 hours (Figure 1). To assess the
irritant potential of the preservatives, IC50 values for each substance
were also calculated, and the results are presented in Table 3. Based
on the IC50 values, the skin irritation potential of the preservatives
was ranked, and phenoxyethanol was the safest among them, with
an IC50 of 2162 μg/mL. Although methylparaben and propylparaben,
the two parabens, ranked second, their IC50 values were one order of
magnitude lower than that of phenoxyethanol. Furthermore, DMDM
hydantoin and imidazolidinyl urea could likely induce skin irritation,
as they both exhibited strong cytotoxicity to NHEK cells, with IC50
TA B L E 3 Results of keratinocyte cytotoxicity
IC50
Substance μg/mL mmol/L
values as low as 53.0 and 61.9 μg/mL, respectively. In summary, the
order of keratinocyte cytotoxicity of the preservatives from low to
high was phenoxyethanol, parabens, and formaldehyde releasers.
3.4 | RBC test: the adverse effect of preservatives
on the cytoplasmic membrane
The RBC test is a cell-based cytotoxicity assay to assess membrane dam-
age of erythrocytes and protein denaturation, which are the initial cellular
responses occurring in ocular irritation. The RBC integrity was examined
by testing cytolysis after adding SDS, which is the internal standard of
the method. In the concentration range of 10 to 60 μg/mL, SDS induced
RBC hemolysis in a dose-dependent manner, and the 50% hemolytic con-
centration of SDS was 28.8 μg/ml (Figure 2A), which was in accord with
the 95% confidence limit of H50 (27.4-30.6 μg/mL) reported by DB-ALM
No. 37.17 At the maximum permissible concentration of each preservative,
phenoxyethanol, methylparaben, imidazolidinyl urea, and DMDM hydan-
toin had negligible hemolytic effects on RBCs (Figure 2B). Nevertheless,
propylparaben caused 100% hemolysis at a concentration of 0.4% (w/v),
which exhibited strong damage to the RBC membrane (Figure 2B).

Phenoxyethanol 2162 15.6

3.5 | HET-CAM: the acute irritancy effect of
Methylparaben 236.4 1.6
preservatives on blood vessels
Propylparaben 174.2 1.0
Imidazolidinyl urea 53.0 0.1
The HET-CAM is an organotypic model for investigating the ad-
DMDM hydantoin 61.9 0.3 verse effects on the fragile blood vessel network of the CAM that

F I G U R E 2 Effect of five preservatives at their regulatory maximum acceptable concentrations on cytoplasmic membrane by RBC test.
Negligible hemolysis (%) far under the irritation cutout of 50% by the preservatives after 10-min exposure indicated no damage of RBC
membrane. (A) Dose-response curve of hemolysis (%) induced by the positive control SDS. (B) RBC test results of five preservatives at the
indicated concentrations. Data were shown as mean ± standard deviation (n = 3)
6 |
(A) (B)
(C) (D)
(E) (F)
(G) (H)
is comparable to the conjunctival responses elicited by irritants.
As all test preservative solutions were transparent, the reaction-
time method was chosen, in which the manifestation of coagula-
tion, hemorrhage, and vessel lysis is recorded in seconds during a
period of 5 minutes. No vascular damage in CAM was observed by
applying 0.9% NaCl and olive oil, both of which were vehicle con-
trols (Figure 3A and B). In contrast, CAM exposure to the positive
control, 0.1 M NaOH, triggered severe hemorrhage and vessel lysis
with an IS of 10.98, that of a severe irritant (Figure 3C and Table 4).
Application of the five preservatives had no visual adverse vascular
alterations on the CAM (Figure 3D-H), and the IS values of these
substances were all 0.07, which was equal to that of the vehicle
controls (Table 4). Therefore, phenoxyethanol, methylparaben,
MA et al.
F I G U R E 3 The acute irritancy effect
of five preservatives at their regulatory
maximum acceptable concentrations
on blood vessels by HET-CAM assay.
Representative images of CAMs after
exposure to A, negative control (0.9%
saline), B, vehicle control (olive oil), C,
positive control (0.1 M NaOH), D, 1%
phenoxyethanol, E, 0.4% methylparaben,
F, 0.4% propylparaben, G, 0.6%
imidazolidinyl urea, and H, 0.6% DMDM
hydantoin
propylparaben, imidazolidinyl urea, and DMDM hydantoin had a
small irritating effect on blood vessels.
4 | D I S CU S S I O N
Preservatives are a kind of indispensable ingredient usually incor-
porated in personal care, household, pharmaceutical, and industrial
products to prevent microbial contamination and to prolong their
shelf life.19 However, preservatives are generally regarded as high-
risk cosmetic constituents, resulting in a market trend of supposed
preservative-free products. Many researchers applied preservatives
at concentrations many times higher than the regulatory acceptable
MA et al.
TA B L E 4 Results of HET-CAM
Substance (concentration)
0.9% NaCl
Olive oil
0.1M NaOH
Phenoxyethanol
(1.0% saline)
Methylparaben
(0.4% olive oil)
Propylparaben
(0.4% olive oil)
Imidazolidinyl urea
(0.6% saline)
DMDM hydantoin
(0.6% saline)
concentrations to perform a patch test on individuals with known
sensitivities or allergies. 20 Apart from human patch tests and rab-
bit Draize tests, few studies have examined the possible mechanism
of the preservative-induced irritation response. Because the preva-
lence explained the information on exposure, we investigated the
most frequently used preservatives in cosmetic products: phenox-
yethanol, methylparaben, propylparaben, imidazolidinyl urea, and
DMDM hyantoin.6,21 The aim of this study was to undertake a pre-
cise and comprehensive evaluation of the skin irritation potential of
five prevalent preservatives in cosmetics by combining patch tests
and multiple alternative methods.
The patch test, which is a diagnostic method for identifying the
cause of allergic contact dermatitis, can also be used to assess the
acute skin irritation potential of cosmetics and topical drugs. 22,23
The ROAT is a use test that mimics daily use of cosmetic products
and can further evaluate the significance of patch test reactions. 24,25
Therefore, combining the patch test and ROAT can provide a com-
plete picture of the skin irritation risk of preservatives. As a skin
safety assessment, the subjects participating in this study were
healthy individuals with no known skin diseases or allergies to cos-
metics, unlike cosmetic contact dermatitis diagnostic studies. The
five preservatives all caused mild erythema on the patch area,
and the ISs were calculated. Propylparaben had the highest score
of 0.13, followed by that of phenoxyethanol, methylparaben, and
DMDM hydantoin of 0.06, and imidazolidinyl urea had the lowest
score of 0.03 (Table 1). Along with this finding, a study comparing
skin irritation of several cosmetic preservatives by a 24-hr patch test
illustrated that methylparaben and phenoxyethanol shared a similar
26
mean IS that was lower than that of propylparaben. In the ROAT,
subjects exhibited noticeable erythema on the application area after
applying imidazolidinyl urea and DMDM hydantoin with mean total
ISs of 0.33 and 0.13, respectively, while phenoxyethanol and the
parabens resulted in no adverse response (Table 2). This observation
was consistent with a previous cumulative irritation test (CIT), which
suggested that the median CIT score of the tested preservatives,
comprising EDTA, DMDM hydantoin, parabens, isothiazolinone,
| 7
Irritation Irritation Vascular responses on
score (IS) category CAMs after treatment
0.07 Nonirritant No visible change
0.07 Nonirritant No visible change
10.98 Severe irritant Hemorrhage and lysis
0.07 Nonirritant No visible change
0.07 Nonirritant No visible change
0.07 Nonirritant No visible change
0.07 Nonirritant No visible change
0.07 Nonirritant No visible change
phenoxyethanol, sorbates, and benzoates, was 0.47%, with most
tests showing none or mild irritation under typical in-use condi-
tions. 27 Although the preservatives displayed no severe reaction,
such as papules or vesicles, the weak erythema response indicated a
skin irritancy potential that might lead to consumer dissatisfaction.
In the experimental situation, all the test preservatives were bother-
some, whereas in the daily use situation, the formaldehyde releasers
had higher irritation potential than phenoxyethanol and parabens.
The results also revealed that the patch test was more sensitive,
time-saving, and cost-effective than the ROAT, and it was suitable
for screening the skin irritation potential of cosmetic ingredients.
Traditionally, rabbit Draize tests, including eye tests and skin
tests, have been used for testing the irritant level of cosmetic,
household, or pharmaceutical products for decades. 28,29 However,
the increasing pressure to mitigate the use of live animals in cos-
metic toxicity testing has led to research into developing alter-
native methods. Consequently, simple and reproducible in vitro
tests based on toxicological mechanisms are becoming essential
for estimating the cutaneous safety of cosmetics. These in vitro
test models consist of erythrocytes, reconstructed human skin,
fertilized egg membrane vasculature, and monolayer-cultured ep-
ithelial cells. 30,31 Keratinocytes are the first living cells exposed
to topically applied cosmetics, so keratinocytes are the most bi-
ologically relevant target for evaluating skin irritation effects.
Wilhelm et al found a significant correlation between the in vitro
keratinocyte cytotoxicity model and the extent of in vivo irritant
dermatitis. 32 We compared the cytotoxicity of the five preser-
vatives on NHEKs by calculating the concentrations resulting in
50% inhibition of cell viability (Table 3). In light of the threshold
value of 1 mmol/L to differentiate cytotoxic and noncytotoxic
substances by Wilhelm et al, 32 phenoxyethanol, methylparaben,
and propylparaben were divided into noncytotoxic chemicals with
IC 50 values equal to or above the cutoff, but imidazolidinyl urea
and DMDM hyantoin were cytotoxic substances with skin irrita-
tion potential (Table 3). This result indicated that the adverse skin
response in the ROAT induced by the two formaldehyde releasers
8 |
might be due to their cytotoxicity to keratinocytes. Mature eryth-
rocytes are a good cell model for studying the cell membrane due
to their lack of nucleus. The initial cellular reactions taking place
in eye irritation procedures are corneal epithelial cell membrane
damage. The RBC test is designed to assess membrane damage,
which is evaluated by the amount of hemoglobin leaking into the
supernatant. 33 Within the test preservatives, only propylparaben
at the regulatory acceptable concentration generated the hemo-
lysis of erythrocytes (Figure 2). This might explain the mechanism
by which propylparaben exhibited the highest IS in the patch test.
Under occlusive conditions, propylparaben might easily penetrate
into the stratum corneum to some extent and then destroy the
epithelial cell membrane, resulting in an irritation response. In ad-
dition, HET-CAM is a versatile and sensitive in vitro assay based
on the response of the CAM, a noninnervated, and highly vascu-
larized membrane, to injury in a similar way as that of mucosal and
subcutaneous tissues. 34 As shown in Figure 3, the five preserva-
tives did not evoke any detectable signs of irritation on the CAM,
which indicated that these test substances had no adverse impact
on the vessels. The symptom of infiltration observed in the patch
test is a vascular reaction in the dermis induced by the irritant.
Because none of the five preservatives was able to alter the sub-
cutaneous vessels, no infiltration was observed in the patch test
and the ROAT.
In conclusion, our study provided evidence that commonly
used cosmetic preservatives have minor skin irritation potential
with mild erythema reactions under acceptable regulatory con-
centrations due to cytotoxicity to keratinocytes and injury to the
cell membrane. Occlusive application in the patch test might have
led to a more sensitive result that all test substances showed weak
erythema reactions, especially propylparaben, due to its abil-
ity to injure the cell membrane. Under typical in-use conditions,
phenoxyethanol, methylparaben, and propylparaben were gentle
to the skin, yet DMDM hyantoin and imidazolidinyl urea had no-
ticeable skin irritation potential according to their cytotoxicity to
keratinocytes. These findings reveal that the combination of in
vivo tests and in vitro assays might offer a comprehensive test
battery to evaluate and compare the cutaneous safety of individ-
ual cosmetic ingredients as well as to understand the underlying
mechanism.
AC K N OW L E D G M E N T S
This work was supported by the Scientific Research Project of
Beijing Educational Committee (KM201810011008) and National
Natural Science Foundation of China (81903361). We thank Qi Liu
for technical advice and Yinggang Xu for technical assistance.
C O N FL I C T O F I N T E R E S T
The authors declared that they have no conflicts of interest to this
work.
ORCID
Yao Pan https://orcid.org/0000-0002-8092-4865
MA et al.
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How to cite this article: Ma X, Wang H, Song Y, Pan Y. Skin
irritation potential of cosmetic preservatives: An exposure-
relevant study. J Cosmet Dermatol. 2020;00:1–9. https://doi.
org/10.1111/jocd.13502