Toxicology in Vitro 25 (2011) 1242–1250

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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

In vitro cytotoxicity and phototoxicity study of cosmetics colorants

K. Tomankova a,⇑, K. Kejlova b, S. Binder a, A. Daskova a, J. Zapletalova a, H. Bendova b, H. Kolarova a,
D. Jirova b
a
Department of Medical Biophysics, Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
b
National Institute of Public Health, Srobarova 48, 100 42 Prague, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the work was early identification of preventable risk factors connected with the consumers
Available online 4 May 2011 usage of products of everyday use, such as cosmetics, toys and children products, and other materials
intended for contact with human skin. The risk factor is represented by substances with irritation poten-
Keywords: tial and subsequent possible sensitisation, resulting in negative impact on human physical and psychical
Colorant health with social and societal consequences. The legislation for cosmetics, chemical substances and
Reactive other products requires for hazard identification the application of alternative toxicological methods
Oxygen species
in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based
Phototoxicity
Comet assay
on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were
employed for identification of early morphological and functional changes on cellular level. Four colo-
rants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert
Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV
irradiation (dose 5 J cm2). Fluorescence methods for the study of cell damage using fluorescence probes
offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellu-
lar production of ROS investigated by molecular probe CM-H2DCFDA, which is primarily sensitive to the
increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level
were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yel-
low soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan
crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell
death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using
Chlorophyllin and Unicert Red K 7054-J.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction by selected coloring agents that are currently allowed in all

This study was focused on colorants widely used in consumer
products, foods and feeds, and thus expected to be safe. Colorants
approved for cosmetics are subject to exclusive regulation in the
Cosmetics Directive 76/768/EEC, Annex IV. However, the available
information on their safety is largely incomplete, sometimes con-
fusing or controversial. The extent of available data frequently does
not comply with requirements of the SCCP’s Notes of Guidance for
the Testing of Cosmetic Ingredients and Their Safety Evaluation
(2006).
Performed experiments aimed to provide missing toxicological
data related to possible phototoxicity and DNA damage induced
Abbreviations: ROS, reactive oxygen species; MMP, mitochondrial membrane
potential; UV, ultraviolet; DNA, deoxyribonucleotic acid; DSU, disc scanning unit;
CI, color index; CAS, chemical association service; PIF, photo irritation factor; MPE,
mean photo effect; SGCE, single-cell gel electrophoresis.
⇑ Corresponding author. Tel.: +420 585 632 103; fax: +420 585 632 167.
E-mail address: katerina.tomankova@upol.cz (K. Tomankova).
cosmetic products, particularly those regularly incorporated in
leave-on products applied on skin areas exposed to sunlight or
coming into contact with mucous membranes. The phototoxic
effects of selected colorants were investigated using the validated
and legally accepted 3T3 NRU Phototoxicity Test (EC, 2008) and a
modified phototoxicity test on NIH3T3 cells with MTT assay as
endpoint. Recently, extensive efforts have been made to develop
in vitro methods for predicting the phototoxicity potential of chem-
icals in order to replace animal testing. Various strategies involving
in vitro tests have been proposed in order to evaluate the photo-
toxic potential of chemicals and drugs. In contrast to the 3T3-NRU
test based on cell monolayer, reconstructed epidermal tissues allow
topical application of a large panel of compounds. Moreover, due to
their 3D structure involving connections between cells and extra
cellular matrix, the presence of a barrier function (stratum
corneum) and by their origin, human keratinocytes, these models
are closer to the target organ, the human skin. Indeed, previous
studies on reconstructed skin models have shown their ability to

0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2011.04.026
 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
confirm or modulate positive outcomes in the 3T3 NRU phototoxic-
ity test (Lelievre et al., 2007).
Ultraviolet light including wavelengths from 280 to 400 nm is the
most damaging radiation to human skin. Following sun exposure,
photoallergy and phototoxicity are the two main drug-photosensi-
tivity reactions. In some cases, photoactivated drugs induce damage
to DNA of skin cells, and this photogenotoxic impact could increase
the risk of skin cancer (Lelievre et al., 2007; Shimoda 1998). UV light
can induce formation of radicals which are able to damage
membranes, DNA and other cellular structures (Sobolev et al.,
2000). Reactive oxygen species (ROS) is a term that has become more
popular because it encompasses other reactive species that are not
true radicals but are nevertheless capable of radical formation in
the intracellular and extracellular environments. Exogenous sources
of ROS include heat, trauma, ultrasound, UV light, ozone, smoking,
exhaust fumes, radiation, infection, excessive exercise and thera-
peutic drugs. On the other hand, endogenous sources are products
of metabolic pathways (electron leakage from mitochondrial
electron transport systems forming superoxide) and functional
generation by host defense (phagocytes) (Chapple and Matthews,
2007; Nordberg and Arńer, 2001).
Upon absorption of UV or visible photons, a photosensitizer can
be promoted to a variety of electronically excited states. However,
the efficiency of the photosensitizing action is generally dependent
on the photophysical properties of the excited triplet state (3Sens),
which is reached via intersystem crossing from the initially formed
excited singlet state (1Sens). The 3Sens species are most often char-
acterized by a lifetime from the microsecond to millisecond range.
Deactivation pathway for 3Sens can be defined as a type I mecha-
nism leading to the generation of radical intermediates which in
turn can undergo further reactions with other substrates, solvent
molecules or oxygen (Amor and Jori, 2000). The process can results
in the formation of oxidized products such as the superoxide anion.
It has a relatively low level of reactivity, however, it can be con-
verted to very reactive and cytotoxic species such as the hydroxyl
radical and hydrogen peroxide. These oxidizing substances have
higher reaction activity than common oxygen molecules (Shimoda
1998). As well known, the ROS can in vivo cause damage to biolog-
ical molecules and induce the emergence of disease (Wang et al.,
2010).
Another deactivation pathway for 3Sens is type II mechanism,
where the energy is transferred to any substrate whose triplet state
energy lies at a lower level compared with the photosensitizer trip-
let state (Inbaraj et al., 2005). Both type I and type II photosensiti-
zation mechanisms generate electrophilic species, hence the most
photosensitive targets are represented by electron-rich biomole-
cules (Amor and Jori, 2000; Shimoda 1998). A critical role is per-
formed by the chemical structure of the photosensitizer,
particularly by its degree of hydrophobicity. Lipophilic dyes are
preferentially associated with the cell membranes; at short incuba-
tion times the plasma membrane represents the main binding site,
while at longer times significant photosensitizer concentrations
are recovered from other subcellular membranes including the
mitochondrial and lysosomal membranes, the golgi apparatus
and the rough endoplasmic reticulum (Amor and Jori, 2000). We
supposed that these mechanisms are applied under UV irradiation
sensitized cells by UV absorbing colorants. All of these dyes require
the presence of molecular oxygen to express their phototoxic ac-
tion (Shimoda 1998).
The oxygen products are capable of causing cellular damage,
ultimately leading to cell death by either necrosis or apoptosis
(Nordberg and Arńer, 2001), which are two distinct types of cell
death that differ in terms of their biochemical and morphological
characteristics as well as their regulatory mechanism. The
subcellular localization of a photosensitizer determines the
efficiency as well as the mechanism of photoinduced cell
1243
inactivation; particularly, it controls the relative weight of apop-
totic and random necrotic pathways which are eventually respon-
sible for cell death (Amor and Jori, 2000). Apoptosis (programmed
cell death) plays an important role in embryogenesis and homeo-
stasis of multicellular organisms. The impairment of the apoptotic
function has been associated with several human diseases. In con-
trast, necrosis (cell lysis) occurs in response to acute and non-
physiological injuries (Yang et al., 2007).
DNA damage can be studied by various methods, for example by
measurement of change of mitochondrial membrane potential
DWm, comet assay or microscopic methods. Mitochondrial dynam-
ics is important factor contributing to apoptotic pathways by
stimulation of caspases and chromosomal fragmentation.
Mitochondria are also the major source to modulate cellular
calcium homeostasis, which is critical for cellular signaling and
chemical cytotoxicity (Chang et al., 2007). The DWm collapse asso-
ciated with the permeabilisation of mitochondrial membrane and
the release of apoptogenic proteins (cytochrome c, AIF, Smac/DIA-
BLO, Endonuclease G, etc.) to the cytosol is one of the typical early
events of a mitochondria-mediated pathway and has been pro-
posed to be the point-of-no-return in many cell death programs
(Krestyn et al., 2010). The comet assay (also called single-cell gel
electrophoresis, SCGE) is a sensitive, simple and quantitative
technique for detection of DNA damage. The comet assay can be
used for the detection of damage such as single and double strand
DNA breaks, oxidative DNA base damage, DNA–DNA/DNA–pro-
tein/DNA-drug crosslinking or DNA repair (Tice et al., 2000; Heaton
et al., 2002). The DNA damage is qualitatively presented by the
amount of the unwound DNA fragments, which resembles a comet,
having a distinct head and tail (Heaton et al., 2002). The head
consists of intact DNA, while the tail is created of broken fragments
of DNA or relaxed chromatin. The amount of DNA damage is
directly proportional to the amount of DNA liberated from the head
(Collins, 2004).
2. Materials and methods
2.1. Tested colorants
The four selected colorants used in cosmetics were P-WS
caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K
7008-J. Information on their color indices (CI), CAS numbers,
synonyms and absorption peaks is given in Table 1.
2.2. Materials and instruments
NIH3T3 (Mouse fibroblast cells) were used as a biological mate-
rial for the MTT viability/phototoxicity test, reactive oxygen species
measurement, comet assay, mitochondrial membrane potential
measurement and apoptotic/necrotic assay. 3T3 Balb/c cells were
used as a biological material for 3T3 Neutral Red Uptake Phototox-
icity. The chemicals used included Dulbecco’s Modified Eagle Med-
ium (DMEM), phosphate buffered saline (PBS, pH 7.4 own
preparation), 5-(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluo-
rescein diacetate (CM-H2DCFDA, Invitrogen Co., USA), 3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,
Sigma Aldrich), Annexin FITC Apoptosis Detection Kit (Sigma
Aldrich), 5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolo-
carbocyanine iodide, 5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethyl-imi-
dacarbocyanine iodide (C25H27Cl4IN4, JC-1, Sigma Aldrich)
dimethyl sulfoxide (DMSO, Sigma Aldrich), HMP agarose (Serva,
Biotech, Czech Republic), LMP agarose (Qbiogene, Genetica, Czech
Republic), Trypsin (Sigma Aldrich), fetal bovine serum (FBS, Sigma
Aldrich), NaCl (Tamda, Czech Republic), EDTA (ethylenediaminetet-
raaceticacid, Lachema, Czech Republic), Tris (tris(hydroxymethyl)
1244 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
Table 1
Coloring agents.
Trade name CI CAS
Caramel P-WS Caramel 8028-89-5
Chlorophyllin 75810 (75815) 85536-03-4
Unicert Red K 7054-J 45410 18472-87-2
Unicert Red K 7008-J 45380 17372-87-1
aminomethane, Sigma Aldrich), Triton X-100 (Serva), NaOH (Sigma
Aldrich) SYBR Green (Invitrogen Co, USA). Measurements were car-
ried out on multi-detection microplate reader Synergy HT, trans-
mission microscope Olympus IX81 with DSU unit (Olympus,
Japan). We used 96 well plates (P-Lab, Czech Republic) for cell lines
cultivation, centrifugal machine (Biotech, Czech Republic), glass
cover slip (P-lab, Czech Republic), electrophoretic tank (Bio-RAD,
Czech Republic), UV light source bank of four Phillips tubes TL-D
18/08 (320–400 nm). UVA meter (Type No. 37, Dr. Hönle, Ger-
many), Phototox Version 2.0 software (ZEBET, Germany). The UV
light source, used also in the ECVAM validation study on 3T3 NRU
PT (SOL 500, Dr. Hönle, Germany), was a doped mercury-metal ha-
lide lamp which simulates the spectral distribution of natural sun-
light (emission spectrum in the range of 280–700 nm) (EC, 2008). A
spectrum almost devoid of UVB (<320 nm) was achieved by filter-
ing with 50% transmission at the wavelength of 335 nm (Filter
H1, Dr. Hönle, Germany).
2.3. Sample preparation
104 cells were incubated in thermobox at 37 °C and 5% CO2
for 24 h in 96-well plates with fresh DMEM. After incubation,
DMEM was replaced by PBS and colorants at concentration
0–1000 lg ml1. Samples were incubated for 1 h in thermobox
and then irradiated by ultraviolet radiation for 50 min with UVA
light intensity 1.7 mW cm2 (total dose 5 J cm2). The emitted en-
ergy was measured before each experiment with a calibrated UVA
meter. Test plates without irradiation were kept 50 min in the dark
at room temperature. After treatment fresh DMEM was added to
the cell culture and the plates were incubated for 6 h in thermobox
at 37 °C and 5% CO2.
2.4. Measurement of reactive oxygen species production
ROS were generated by colorants in concentrations between 0
(control group) and 1000 lg ml1. Immediately after UVA irradia-
tion the ROS production was determined using CM-H2DCFDA
fluorescence probes and microplate reader Synergy HT. Time of
CM-H2DCFDA probes incubation was 30 min.
2.5. MTT viability/phototoxicity test
The cytotoxic/phototoxic effect of the colorants on NIH3T3 cells
was determined using the MTT assay. We replaced DMEM by PBS
prior to starting the MTT measurements, added 20 ll of 20 mM
MTT (dissolved in PBS) and incubated the cells for 3 h at 37 °C
and 5% CO2. The MTT solution was carefully removed and 100 ll
DMSO was added in order to solubilize the violet formazan crys-
tals. Data were calculated using the Phototox software.
2.6. Apoptosis/necrosis assay
We replaced DMEM by Binding buffer 1 before starting the
measurements, added 5 ll of propidium iodide and 2.5 ll of
annexin incubated at 37 °C and 5% CO2 for 30 min and then washed
the cells by Binding buffer. The annexin and propidium iodide
Synonyms Absorption (nm)
E150 282
E140, E141 340, 503 and 656
Acid Red 92 295, 309, 344, 360, 511
Eosin Y, Acid Red 87 338, 298, 343, 492
fluorescence signals were recorded by fluorescence microscope
with CCD camera, manually scored and counted as percentage of
control group (100  average of test group/average of control
group).
2.7. Mitochondrial membrane potential assay DWm
Mitochondrial membrane potential (MMP) was monitored by
the fluorescent cationic voltage-dependent dye JC-1. NIH3T3 cells
were loaded in PBS media with JC-1 (5 lg ml1, dissolved in
DMSO), for 20 min at 37 °C, 5% CO2 and then washed by PBS.
Results were expressed as the fluorescence retained within the
cells.
2.8. Comet assay
Microscope slides were first precoated with 1% HMP agarose in
distilled H2O and then placed in a drying oven at a temperature of
60 °C for at least 30 min. Then 85 ll of 1% HMP agarose in PBS was
applied onto the recoated slides which were then covered with a
cover slip. The slides were placed in a refrigerator in order to
enhance gelling of the agarose. The cells were trypsinized for
5 min and trypsinisation was stopped by means of fetal bovine
serum (FBS). Isolated cells were centrifuged (6 min, 1000 rpm),
then the cell pellet was dispersed in 20 ll of PBS and vortexed. A
quantity of 85 ll of 1% LMP agarose was added to this solution
and 85 ll of this suspension was added to the microscope slide
that had been prepared with the solidified agarose gel (the cover
slip was removed prior to cell inoculation on the gel), covered by
a new glass cover slip to form a thin layer and moved to the refrig-
erator again. After solidifying the cover slips were removed again
and the microscope slides were immersed in a lysis buffer contain-
ing 2.5 M NaCl, 100 mM EDTA 10 mM Tris (tris(hydroxy-
methyl)aminomethane), 1% Triton X-100, pH = 10) at 4 °C for at
least 1 h. After the lysis the slides were washed in distilled water
to remove all salts, then placed in an electrophoretic tank and
dipped into a cool electrophoresis solution (300 mM NaOH,
1 mM EDTA) for 40 min. Electrophoresis was run at 0.8 V.cm1 and
380 mA for 20 min. After the electrophoresis the slides were rinsed
3 times for 5 min with a neutralisation buffer (0.4 M Tris, pH = 7.5)
at 4 °C. The samples were subsequently stained by SYBR Green and
immediately scored. Cells were analysed using a fluorescence
microscope with CCD camera and manually scored. We evaluated
randomly chosen 100 cells from each sample. The cells were
classified in five classes, i.e., 0, 1, 2, 3, 4 according to the length
of their tail determining the degree of the DNA damage.
3. 3T3 Neutral Red Uptake Phototoxicity
The test was performed according to INVITTOX Protocol No. 78
(http://ecvam-dbalm.jrc.ec.europa.eu), using 3T3 Balb/c fibro-
blasts, passage 75–85. A test substance is predicted as having a
potential phototoxic hazard if the photoirritation factor (PIF), cal-
culated as the ratio of toxicity for each substance with and without
UV light, is higher than five. Using the Phototox software, a second
predictor of phototoxicity, the mean photo effect (MPE) was also
 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
calculated. The MPE is a statistical comparison of the
dose–response curves obtained with and without UV and a test
substance is predicted as phototoxic if MPE is higher than 0.1.
According to the Commission Regulation No.440/2008, a test sub-
stance with a PIF > 2 and < 5 or an MPE > 0.1 and < 0.15 is predicted
as ‘‘probably phototoxic’’ (EC, 2008).
3.1. Fluorescence microscopy
Cell viability and morphological changes were visualized by
Olympus IX80 microscope with DSU unit. Images were recorded
by CCD camera and CellR software.
3.2. Statistical analysis
The results were processed using software SPSS v. 15 (SPSS Inc.
Chicago, USA). The data are presented as mean ± SD of three inde-
pendent experiments. The statistical significance was determined
by an analysis of variance with ANOVA post hoc tests Dunnet
(comparison with control). The statistical analysis of the results
of apoptotic/necrotic assay and comet assay was performed using
Fisher exact tests with Bonferroni correction. P 6 0.05 was consid-
ered statistically significant.
4. Results
All substances were tested for their phototoxicity potential
using a battery of in vitro tests. The effect of colorants concentration
on ROS formation in mouse fibroblast cells was monitored with and
without UV light irradiation up to the dose of 5 J cm2. We observed
an increase in ROS production immediately after completion of 1 h
incubation and 50 min irradiation (+UV group) or dark incubation
(UV group) of cells with the colorants. The summary of the values
for colorants concentrations (0–1000 lg ml1) are presented in
Fig. 1. The obtained data showed difference in ROS production
Fig. 1. Reactive oxygen species production elicited by 4 cosmetics colorants with (+UV) and without irradiation (UV) immediately after therapy. The ROS production of the
samples was determined using relative fluorescence units (RFU). Significant results corresponding to control are marked with asterisks.
1245
between the control cells and cells exposed to colorants with
(+UV, dark grey columns) and without (UV, light grey columns)
ultraviolet light application. The of ROS production in control
groups +UV is 8-fold higher than the ROS production of control
UV (see Fig. 1). The MTT assay supports these results as the cell
viability after +UV treatment was decreased about 10% in compar-
ison with control –UV (data not shown). In comparison with the
control group + UV (without colorants), ROS production in + UV
irradiated cells was significantly higher in the presence of P-WS
Caramel, at concentrations 865.7 and 1000 lg ml1. Significant in-
creases of ROS production were detected in Unicert Red K 7054-
J + UV in concentration 111.1–1000 lg ml1 and UV in concentra-
tions 10.1 and 1000 lg ml1. Unicert Red K 7008-J induced signifi-
cant increase in in concentrations 1.1, 2.7, 8.7, 48.7 and
1000 lg ml1 (+UV) and in concentrations 10.1–48.7 lg ml1
(UV). In conclusion, significantly augmented production of ROS
was detected in case higher concentrations of the tested colorants
after UV irradiation, while without irradiance treatment and over
significant results were detected only in case of Unicert Red K
7008-J. Chlorophyllin did not induce any significant increase in
ROS production.
The 3T3 Neutral Red Uptake Phototoxicity test revealed photo-
toxic potential of all the four tested colorants. However, the photo-
toxic effect of P-WS Caramel was induced only in very high
concentrations (mean EC50 (+UV) = 760.5 lg ml1). The MTT
phototoxicity test classified Unicert Red K 7054-J as probably pho-
totoxic (PIF = 2.1) and Chlorophyllin and Unicert Red K 7008-J as
phototoxic (Table 2).
Another early effect of colorants activity in the investigated cell
lines was depolarization of the mitochondrial membrane. The col-
orants may increase the level of ROS, leading to mitochondrial
damage in the investigated cell lines. In order to monitor DWm,
we chose the JC-1 fluorescent dye which has been demonstrated
to be more specific for mitochondria than other fluorescent dyes
as DiOC6 or Rhodamine 123 (Rogalska et al., 2008). The MMP assay
was used to evaluate the DWm changes in cells 6 h after treatment.
1246 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
Table 2
Phototoxicity tests.
Coloring agent 3T3 NRU PT NIH3T3 MTT PT
PIF MPE PIF
P-WS Caramel 6.3 0.282 1.3
Chlorophyllin 82.1 0.407 12.0
Unicert Red K7054-J 22.6 0.441 2.1
Unicert Red K7008-J 170.2 0.458 232.2
The results of the JC-1 fluorescence assay showed a concentration-
dependent decrease in mitochondrial membrane potential of the
studied cells. Depolarization of mitochondrial membrane
predominated in higher concentrations of the colorants
(273.9–1000 lg ml1) regardless of + UV or UV treatments with
the exception of Unicert Red K 7008-J and Chlorophyllin, where
the higher decrease without irradiation (UV) than after +UV
treatment was recorded (Fig. 2). Particularly in case of Chlorophyl-
lin, significant large decreases in 41.7 and 865.7 lg ml1 concen-
trations UV were observed.
Microscopy using fluorescence dyes, annexin and propidium
iodide, was used for viability analysis of NIH3T3 cells treated by
coloring agents with and without UV irradiation with light dose
5 J cm2 (Fig. 3). The method revealed significantly increased
number of apoptotic and necrotic cells +UV and UV in case of
Unicert Red K 7054-J in all tested concentrations (highest concen-
trations were not available due to the cytotoxicity of the colorant).
Application of P-WS Caramel significantly induced apoptotic
change in all concentrations in +UV and UV. However, the in-
creased numbers of necrotic cells were detected only in concentra-
tions + UV 273.9–1000 lg ml1 and UV 865.7–1000 lg ml1.
Significant number of apoptotic cells was observed in case of Chlo-
rophyllin + UV in all concentrations (as well as Unicert Red K 7008-
J), in –UV only in concentration 20.4 lg ml1 (Unicert Red K 7008-J
in concentrations 0.2, 2.7 and 48.7 lg ml1).
The results of the comet assay are shown in Table 3. The cells
were classified in 5 classes, 0, 1, 2, 3, 4, according to the length
Fig. 2. Cell mitochondrial membrane potential 6 h after therapy (+UV) and without irradiation (UV). The MMP of the samples was determined using relative fluorescence
units (RFU). Significant results corresponding to control are marked with asterisks.
of their tail determining the degree of the DNA damage. The
highest proportion of damaged cells (class 3 and 4) was deter-
mined in case of Chlorophyllin + UV, Unicert Red K 7008-J + UV
MPE and Unicert Red K 7054-J in all concentrations and treat-
ments + UV and -UV. No intense comets were detected in the
0.036
0.111
exposed control.
0.116
0.351
5. Discussion
Colorants of Column 1, Annex IV, to the Cosmetics Directive are
currently allowed in all cosmetic products including leave-on
products. Available toxicological information often comprises only
selected exposure conditions (e.g. for hair dyes) and does not
reflect all foreseeable conditions of use that may lead to relatively
high exposure (SCCNFP/0803/04 2004). Colorants appear in formu-
lations of hair dyes or decorative cosmetics applied to body parts
exposed to sunlight. Despite being obvious photoabsorbers, they
are not regularly tested for phototoxicity. This paper was aimed
at filling the considerable gaps in knowledge on colorants used in
cosmetics, foods and feeds. Toxicological hazard of phototoxic
effects of these substances is not addressed properly in any of
the available sources of scientific information (e.g. HSDB – Hazard-
ous Substances Data Bank, IUCLID Dataset, RTECS – Registry of
Toxic Effects of Chemical Substances, opinions of the Scientific
Committee on Consumer Safety, EC). However, the use of photoac-
tivated chlorophyllin-based gelatin films to prevent microbial
contamination of food products (López-Carballo et al., 2008) or
the application of xanthenes and porphyrins as sunlight-activated
insecticides (Amor and Jori, 2000) suggest phototoxic potential of
colorants, which might be responsible for adverse reactions to
cosmetics (Wolf et al., 2001). The synergistic action of cosmetic
products, namely lipsticks, and sunlight, described by Hanson
et al. (2008) might be attributed to the colorants present in the
formulations.
We proved photodynamic effects in vitro for all four tested
colorants. The cytotoxicity of the colorants was accompanied by
an increase in production of reactive oxygen species and
 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
Fig. 3. Percentage of apoptotic and necrotic cells after exposure to 4 cosmetics colorants with (+UV) and without irradiation (UV) 6 h after therapy. Significant results to
correspond control are marked with asteriks.
depolarization of mitochondrial membrane potential as indicated
in Figs. 1 and 2. Three of the substances exhibited concordant re-
sults in both in vitro phototoxicity tests. Discrepancy ocurred only
in case of P-WS Caramel, classified as phototoxic in 3T3 NRU PT
and non-phototoxic in NIH 3T3 MTT PT, however, this substance
elicited phototoxicity in 3T3 NRU PT only in high concentrations,
i.e. is probably false positive. The other colorants were classified
as phototoxic or probably phototoxic in both tests. The degree of
toxic response might have been affected by the use of two vital
dyes with different mechanisms of cell viability assessment, how-
ever, both tests revealed the phototoxic potential of Chlorophyllin,
Unicert Red K7054-J and Unicert Red K7008-J.
Oxidative stress can cause change of DWm, cell aging or cell
death (Valdez et al., 2006; Chang et al., 2007). ROS production
and mitochondrial dysfunction are therefore the possible contrib-
uting factors of colorants toxicity. Stressed cells may produce
ROS and particularly UV irradiation has great influence on ROS
production as can be seen in differences between contol groups
+UV and UV (see Fig. 1). ROS amount is dependent upon the
length of time of UV irradiation (Hanson et al., 2006). Fluorescence
probe CM-H2DCFDA can reveal the presence of H2O2 (hydrogen
peroxide), OH (hydroxyl radical), COO (peroxyl radical) and HOCl
(hypochlorous acid). Several quenchers can be used to determine
the kind of generated ROS, for example sodium azide (NaN3) and
histidine can quench the singlet state molecular oxygen (1O2),
while 2,6-di-tert-butyl-methylphenol and mannitol can quench
the hydroxyl radicals (OH). And that, the vitamin C can quench
almost all kinds of ROS. Wang et al. 2010 revealed that in presence
of Chlorophyllin-Metal complex, under visible-light irradiation,
several kinds of ROS can be generated, at least including 1O2 and
OH. Chignell and Sik, 2003 observed dose-dependent increase in
fluorescence signal in cells loaded with 20 ,70 -dichlorofluorescin
after UVA irradiation. Fluorescence in cells exposed to 4 J cm2 of
UVA exhibiting an almost 10-fold increase over dark controls. This
statement supports our results as we observed 8-fold increase in
the ROS production over dark control after UV irradiation at dose
5 J cm2 (see Fig. 1). Our results revealed that cell viability in
1247
control group after UV irradiation at dose 5 J cm2 did not excess
90% in comparison with the dark control (100%) (data not shown).
On the other hand, Inbaraj et al. and Chignell and Sik, 2003 claimed
that UVA at 4 J cm2 had no effect on the viability of cells. (Inbaraj
et al., 2005; Chignell and Sik, 2003).
It has been suggested that changes of mitochondrial membrane
potential play a key role in apoptotic cascade and the DWm
changes can reveal an early stage of apoptosis. A decrease of
mitochondrial membrane potential has been hypothesized to be
a marker of apoptotic cells (Matarrese et al., 2003) and ROS
production is preceded by a decrease in mitochondrial membrane
potential (Rogalska et al., 2008; Chang et al., 2007)). Our results
showed decreasing of DWm in dependence on the applied concen-
tration of colorants and UV irradiation (see Fig. 3). Depolarisation
of mitochondrial membrane potential is an implication of perme-
ability transitive pores opening or damage of the outer mitochon-
drial membrane integrity. Matarrese et al. studied HIV-infected
cells, and hyperpolarization of MMP was observed. They also
suggest that HIV protease inhibitors, by interfering with induction
of the mitochondrial hyperpolarization state, can result in cell
survival even independent of any viral infection (Matarrese et al.,
2003). No significant mitochondrial membrane hyperpolarization
was observed in our experiment.
Specific chemicals, drugs and colorants can induce ROS medi-
ated apoptosis and necrosis pathways in all cell lines depending
on the length of the post-treatment time (Rogalska et al., 2008).
Under our experimental conditions we have shown that colorants
(particularly Chlorophyllin, Unicert Red K 7054-J and Unicert Red K
7008-J) can induce apoptosis and necrosis in NIH3T3 cells.
Fragmentation of DNA by endonucleases is an indicator of cellular
apoptosis (Chang et al., 2007; Sun and Liu, 2006). Our observations
suggest that colorants may induce both necrosis and apoptosis in
NIH3T3 cells. P-WS Caramel and Unicert Red K 7054-J elicited
significant apoptosis and necrosis both after +UV and without
UV irradiation. On the other hand, Unicert Red K 7008-J and Chlo-
rophyllin induced apoptosis after +UV irradiation in all the tested
concentrations (see Fig. 3).
1248 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250

Table 3
Percentage of cells distributed to 5 classes of DNA damage determined by comet assay with (+UV) and without irradiation (UV) 6 h after therapy. Significant results
corresponding to control are typed in italics.

P-WS Caramel +UV (lg ml1)

Comet class 1000 865.7 273.9 111.1 48.7 22 10.1 4.6 2.2 Control
0 19 93
1 14 20 30 20 70 59 70 75 7
2 30 5 34 10 20 31 30 6 0
3 14 35 30 36 60 6 8 0
4 86 21 45 10 4 2 0
P-WS Caramel UV (lg ml1)
0 69 85 85 74 82 81 82 84 84 92
1 20 8 10 5 8 10 11 13 13 6
2 11 3 5 4 3 6 5 4 2 1
3 5 8 3 5 1 1
4 0
Chlorophyllin +UV (lg ml1)
Comet class 1000 865.7 273.9 111.1 41.7 20.4 10.1 8.7 4.7 2.7 2.1 1 0.9 Control
0 37 95
1 55 5
2 1 3 6 0
3 1 2 5 6 14 16 3 0
4 99 98 95 94 86 81 0
Chlorophyllin UV (lg ml1)
0 13 85 85 86 85 94 94
1 14 42 82 15 15 14 14 6 6
2 86 58 5 1 0
3 0
4 0
Unicert Red K 7054-J +UV (lg ml1)
Comet class 1000 865.7 273.9 111.1 48.7 22 10.1 4.6 Control
0 5 10 96
1 69 81 4
2 1 26 8 0
3 1 1 4 3 5 3 0
4 99 99 96 97 95 96 0
Unicert Red K 7054-J UV (lg ml1)
0 27 96
1 72 4
2 1 0
3 1 1 2 2 3 0
4 100 100 99 99 98 98 97 0
Unicert Red K 7008-J +UV (lg ml1)
Comet class 1000 111.1 48.7 22 10.1 8.7 2.7 1.1 0.5 0.2 Control
0 49 69 25 15 95
1 8 89 51 26 75 80 5
2 7 67 11 5 5 0
3 4 30 25 0
4 96 63 0
Unicert Red K 7008-J UV (lg ml1)
0 2 5 1 4 11 10 33 92
1 95 93 92 96 96 89 89 67 8
2 15 5 3 3 1 0
3 0
4 0

The degree of DNA damage in the comet assay can be assessed
either automatically using appropriate scoring software or manu-
ally. Recent research proved that the visual scoring produces the
same results compared to the computerized image analysis (García
et al., 2004). The data obtained from the visual scoring highly
correlate with the quantitative parameters commonly used in the
comet assay (tail moment, tail length) (Heaton et al., 2002). The
extent of DNA damage was determined according to the length
of a tail of the comet determining the degree of the DNA damage
(Collins, 2004). The chosen cells treated with each sample were
visually analyzed and divided in five classes (0–4), where 0 refers
to undamaged cells and comet class 1 to minimally damaged cells
(Vanzella et al., 2007). DNA damage can be considered as medium
when comet class 2 is prevailing. Comet class 3 corresponds to
large DNA damage and cells assigned comet class 4 refer to
apoptotis (Santos et al., 2010). UV exposure may affect diverse bio-
logical functions including DNA replication, repair, cell cycle con-
trol and chromatin remodelling (dos Santos Montagner et al.,
2010). DNA repair mechanisms are involved immediatelly after
UV exposure and complete DNA recovery takes about 2 h (de
Width and Greulich, 1994). To avoid any mechanisms involved in
DNA reconstruction we performed the comet assay in 6 h after
UV irradiation to assure that our results indicate irreversible DNA
damage leading to cell death.
 K. Tomankova et al. / Toxicology in Vitro 25 (2011) 1242–1250
In summary, three of the four tested colorants exhibited photo-
dynamic properties leading to ROS production, phototoxic effects
and DNA damage. Moreover, colorant Unicert Red K 7054-J is
potentionally mutagenic/genotoxic regardless of photoactivation.
The information in literature about these four tested colorants is
scarce, incomplete and often controversial. They are used in a wide
variety of cosmetics, from hair dyes, shampoos, powders to
lipsticks. A literature research has shown, that particularly the
colorants Unicert Red K 7054-J and Unicert Red K 7008-J represent
ingredients found in leave-on cosmetic products as make-up foun-
dations, lipsticks, face creams, even sunscreen products, intended
for use on body parts exposed to sunlight. Our positive findings
suggest that colorants exhibiting phototoxicity and/or mutagenic-
ity potential, may contribute to adverse skin reactions and body
systems diseases, e.g., phototoxicity, photoallergy or even
(photo)carcinogenicity (namely Unicert Red K 7054-J and Unicert
Red K 7008-J). The concentration recommended by manufacturer
for Unicert Red K 7008-J is up to 0.5%, however, Annex IV of the
Cosmetic Directive does not regulate any of these colorants. In
some cosmetic products, e.g., body paints for children, the concen-
tration of colorants in formulation may reach up to 5%.
However, results of the tests in vitro may not be directly related
to human reaction in vivo as the bioavailability may differ (Jirova
et al., 2010) and other toxicological data should be obtained, par-
ticularly information on skin penetration/absorption. We at-
tempted to estimate the possible exposure scenarios when these
colorants are present in cosmetic formulations in relation to the
concentrations used in the in vitro studies. We considered the
exposure scenario for lipsticks where both red colorants are regu-
larly present. The penetrated dose in lips may reach 285 lg per
day. The value may be derived from the presumption that colo-
rants are present in cosmetic formulations up to 5%. The calcula-
tion for Unicert Red K 7054-J, which is phototoxic and positive in
Comet assay regardless of irradiation, may be as follows: The esti-
mated applied daily amount of a lipstick reaching 0057 g, 5% con-
tent of colorant in formulation, considered dermal absorption of
10% (MW = 829,64) leads to an estimated dose of 285 lg per day
(SCCS, 2010). In the Comet assay, the DNA damage effect was found
when cells were exposed to the dose of 1 lg (concentration of
10 lg ml1 in 0.1 ml per well). The phototoxic effect in the 3T3
NRU PT was detected when cells were exposed to a dose of about
0,8 lg (EC50 (+UV) 0.827, resp. 0.711 lg ml1, in 0.1 ml per well,
data not shown). Although the phototoxic effect and DNA damage
may not be immediately apparent under normal conditions of use
because of subclinical reaction, possibly masked by the presence of
other ingredients in the product formulation, the cumulative sub-
clinical effect may contribute to delayed adverse changes of the
skin tissue. Moreover, it is known that a number of photosensitiz-
ing dyes have no phototoxic effect when applied topically to intact
skin, however, elicit phototoxic reactions when applied to chaffed
or damaged skin (Morikawa et al., 1976.
In conclusion, missing toxicity data for these four substances
and a number of other colorants allowed in all cosmetics should
be completed so that their possible hazard can be taken into
account when safety assessment of finished cosmetic formulations
is performed. Our findings suggest that concern is warranted about
the use of these colorants in cosmetic products, as a food additive
or in insecticidal sprays.
Acknowledgement
This work was supported by the Ministry of Health NS9648-4/
2008, Ministry of Education of the Czech Republic MSM
6198959216, GACR 202/09/1151, GACR P304/10/1316, LF 2011/
009 and GACR 303/09 H048 and Institute of Molecular and Trans-
lational Medicine CZ.1.05/2.1.00/01.0030.
1249
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