Embryotoxicity of Cosmetic Compounds Assessed by Embryonic Stem Cell Test

Introduction
Cosmetics are closely related to human daily life, and the
identification of embryotoxic potentials of compounds in
cosmetic products is imperative. However, in vivo animal
tests are mostly time-consuming, expensive, and require
large numbers of animals. In 2003, the Seventh Amendment
to the Cosmetics Directive setting a deadline for animal
tests performed in the cosmetic industry was endorsed
by the EU Cosmetics Committee. The key strengths of the
Seventh Amendment to the Cosmetics Directive included:
(1) A phased ban on animal testing of cosmetics to come
into effect from 2009, for tests where non-animal alterna-
tives exist. (2) A total ban on animal testing of cosmetics,
to come into effect from 2013, whether or not non-animal
alternatives are available. To meet the requirement of the
amendment, there is a strong demand to find a useful tool
for in vitro embryotoxic tests of cosmetic products.
Three in vitro embryotoxic tests were validated by the
European Centre for the Validation of Alternative Methods
(ECVAM), including the embryonic stem cell test (EST),
the micromass test (MM), and the whole embryo culture
assay (WEC) (Genschow et al. 2000; 2004; Piersma et al.
2004; Spielmann et al. 2004). A set of 20 test compounds
with known in vivo embryotoxicity were tested under blind
conditions in different laboratories. The accuracy of the
EST, the MM, and the WEC assay was 78%, 70%, and 80%,
respectively. The predictivity was 100% for each approach
to classify strong embryotoxic compounds. For the MM and
WEC tests, pregnant animals still need to be sacrificed to get
the embryos. While in the EST, only established cell lines are
(Received 02 November 2009; revised 22 December 2009; accepted 23 December 2009)
ISSN 1537-6516 print/ISSN 1537-6524 online © 2010 Informa UK Ltd
DOI: 10.3109/15376510903585450 http://www.informahealthcare.com/txm
RESEARCH ARTICLE
Assessment of embryotoxicity of compounds in cosmetics
by the embryonic stem cell test
Rui Chen1,2
, Jing Chen1,2
, Shujun Cheng3
, Jie Qin1,2
, Weiqiang Li1,2
, Lirong Zhang4
, Hong Jiao3
, Xinbing
Yu1,2
, Xiuming Zhang1,2
, Bruce T. Lahn1,2
, and Andy Peng Xiang1,2
1
Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, 74#
Zhongshan Road 2, Guangzhou,
Guangdong. PR China 510080, 2
The Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education,
Guangzhou 510080, PR China, 3
Food Lab of Guangdong Entry-Exit Inspection and Quarantine Technology Center.
Guangzhou 510623, PR China, and 4
Department of Pathophysiology, Guangdong College of Pharmacy, Guangzhou
510006, PR China
Abstract
The new EU legislations for cosmetics (Seventh Amendment) have laid down deadlines for the replacement of
animal tests in cosmetics.This policy stimulates the acceptance of in vitro approaches to test embryotoxic poten-
tials of compounds in cosmetics products. The embryonic stem cell test (EST) designed by The European Centre
for the Validation of Alternative Methods (ECVAM) is currently the most promising in vitro assay to predict the
embryotoxic potential of compounds. In this study, six selected compounds (hydroquinone, eugenol, dibutyl
phthalate, antimony (III) oxide, neodymium (III) nitrate hydrate, melamine) formerly involved in cosmetic products
were selected to test their embryotoxic potentials by the EST. 5-Fluorouracil and penicillin G were separately set
as positive and negative control.The embryotoxic potential was determined by the prediction model (PM), which
was calculated from three endpoints, the IC50
3T3, IC50
ES, and ID50
. Hydroquinone, eugenol, and antimony (III) oxide
indicated with strong embryotoxicity, while dibutyl phthalate, neodymium (III) nitrate hydrate, and melamine
exhibited a weak embryotoxicity. These results may provide a valuable attempt to expand the application of EST
to the cosmetics field.
Keywords: Cosmetics; embryotoxicity; embryonic stem cell test
Toxicology Mechanisms and Methods, 2010, 1–7, Early Online
Address for Correspondence: Andy Peng Xiang, Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, 74#
Zhongshan Road 2, Guangzhou,
Guangdong, 510-080, PR China. Tel: +8620-87335822. Fax: +8620-87335858. E-mail: xiangp@mail.sysu.edu.cn
ToxicologyMechanismsandMethodsDownloadedfrominformahealthcare.combyUniversityofChicagoLibrary
Forpersonaluseonly.

2 Rui Chen et al.
needed. Therefore, the EST is currently the most promising
in vitro alternative to the in vivo animal tests.
The EST was first established in 1997 by Spielmann et al.
(1997). Two permanent mouse cell lines (ES cell line D3, 3T3
fibroblasts)wereusedintheEST.Thedifferentiationprocedure
of ES cells in vitro is similar to the development of embryo in
vivo. To this point, ES cells are suitable to evaluate the embryo-
toxic potential of compounds in vitro (Rohwedel et al. 2001).
Three end-points were obtained from concentration–response
curves, including (1) the concentration of the tested com-
pounds causes 50% inhibition of differentiation of ES cells
into cardiomyocytes (ID50
); and (2) the concentration of the
tested compounds that inhibits 50% viability of ES cells (IC50
ES) or 3T3 fibroblasts (IC50
3T3). By the prediction model
(PM) (Spielmann et al. 1997; Scholz et al. 1999) based on the
three endpoints, tested compounds were classified into three
classes: strongly, weakly, or non-embryotoxic.
EST has got global attention since it was established
and validated by the ECVAM. It was applied to screen the
embryotoxicity of chemicals in pharmaceuticals, of metals
for medical use, and chemicals in other fields (Imai and
Nakamura 2006; Paquette et al. 2008; Stummann et al. 2008).
Furthermore, new protocols have been developed to improve
the practicability and predictivity of EST, including a high
throughput screening method (Peters et al. 2008a; b), and
additional endpoints of cellular differentiation (zur Nieden
et al. 2004; Festag et al. 2007).
In the current study, we reported to test the embryotoxic-
ity of six compounds formerly involved in cosmetics by the
EST for the first time. Among the six selected compounds,
hydroquinone, eugenol, dibutyl phthalate, and antimony (III)
oxide were once used as ingredients in cosmetics, and now
are forbidden or limited in cosmetics. Hydroquinone had
been used in commercially available over-the-counter skin
lightener products and was forbidden after 2001. Eugenol is
a fragrance compound added into perfume, but the content
is strictly controlled. Dibutyl phthalate had been added into
nail polish in cosmetics, and was banned for use in almost all
countries. Antimony is a kind of heavy metal and had been
used as rouge and black paint for eyebrows and was pro-
hibited in cosmetics. Melamine and neodymium (III) nitrate
hexahydrate were wildly applied in other industries and both
were once found to contaminate cosmetic products. Three
endpoints (IC50
3T3, IC50
ES, and ID50
) of each test compound
were obtained, and each test compound was classified to be
strongly, weakly, or non-embryotoxic.
Materials and methods
Cell culture
Murine ES cell line E14 (ATCC No: CRL 1821, USA) and
Balb/c 3T3 (ATCC No: CCL-163, USA) cells were used in this
study. Two permanent cell lines were cultured at 37°C and
5% CO2
and routinely passaged three times a week. E14 cells
were cultured on mouse embryonic fibroblast feeders (MEF)
in a standard ES-cell culture medium containing DMEM
(Invitrogen, USA) supplemented with 10% FCS (Hyclone,
USA) 2 mM glutamine (Sigma, USA) 1% non-essential amino
acids (Sigma), 0.1 mM b-mercaptoethanol (Merck, Germany)
1000 U/ml leukemia inhibiting factor (Chemicon, USA) 50 U/
ml penicillin G, and 50 µg/ml streptomycin (Sigma). 3T3
fibroblasts were cultured in DMEM (Invitrogen) containing
10% FCS (Hyclone), 4 mM glutamine (Sigma), 50 U/ml peni-
cillin G, and 50 µg/ml streptomycin (Sigma).
Cytotoxicity assay
To determine cytotoxic effects of the tested compounds on
3T3 cells and E14 cells, the MTT cytotoxicity assays were per-
formed as previously described (Spielmann et al. 1997; Scholz
et al. 1999). In brief, on day 0, 500 cells in 50 µl routine culture
medium without LIF were seeded into each well of a 96-well
plate (Corning, USA). Culture medium (150 µl) with or without
tested compounds were added to each well after 2 h incuba-
tion at 37°C and 5% CO2
. Each test concentration and positive
control was tested in six replicated wells, and seven concentra-
tions were set for each compound in 1:10 dilution. On days 3
and 5, medium was changed with fresh medium containing
the respective concentration of tested compounds as on day
0. On day 10, a MTT assay (Mosmann 1983) was performed to
determinecellviability;20µl5 mg/mlMTT(3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazoliumbromide;Sigma)dissolved
in PBS was added into each well. After incubation at 37°C and
5% CO2
for 2 h, the medium was removed. Cells were incu-
bated with 130 µl MTT desorbing solution containing 3.49%
(v/v) of 20% SDS stock solution and 96.51% (v/v) 2-propanol.
Absorbancewasmeasuredbyamicroplatespectrophotometer
(Tecan, Germany) at 570/630 nm. The cytotoxicity of the tested
compounds was determined from the inhibiting concentra-
tion of 50% (IC50
) obtained through concentration–response
curve.
Differentiation assay with ES cells
To detect the effects of tested compounds on the differentia-
tionofEScellsintocardiomyoctyes,differentiationassayswere
done as previously described (Spielmann et al. 1997; Scholz
et al. 1999). Briefly, 750 cells in 20 µl cell culture medium with-
out LIF were seeded as droplet into the lid of a 10-cm Petri
dish (Corning), filled with 10 ml PBS and incubated at 37°C
and 5% CO2
. After 3 days of hanging drops culture, the cells
formed aggregates called embryonic bodies (EBs). The EBs
were transferred into a 6-cm bacterial Petri dish (Corning)
and cultured in suspension for 2 days. On day 5, the EBs were
placed separately into 24-well plates (Corning) containing the
appropriated concentration tested compounds, and allowed
to attach and outgrow for an additional 5 days. On day 10,
the number of wells containing beating myocardial cell areas
was counted by using a phase-contrast microscope. Besides
solvent control, seven concentrations were set for each com-
pound in 1:10 dilution from 1 mg/ml, except penicillin G from
10 mg/ml and antimony (III) oxide from 10 µg/ml. One 24-well
plate containing 24 EBs was used for one test concentration
and solvent control. An assay was valid when the solvent con-
trol plate had at least 21 wells out of 24 that contained beating
myocardial cell areas, and this number were set as 100%. The
Forpersonaluseonly.

Cosmetics embryotoxicity and in vitro test 3
inhibition of differentiation (ID50
) was calculated from the
concentration–response curve.
Tested compounds
5-Fluorouracil (Sigma, CAS No. 51-21-8) with a known strong
embryotoxicity was selected as a positive control. Penicillin G
(Sigma, CAS No. 69-57-8) was used as a negative control.
Two compounds were both dissolved in PBS. Six tested com-
pounds except Antimony (III) oxide were purchased from Alfa
(Germany) (Table 1). Stock solutions of hydroquinone (CAS
No.123-31-9)andmelamine(CASNo.108-78-1)wereprepared
in DMEM, and hydroquinone was light protected through the
whole experiment. Eugenol (CAS No. 97-53-0) and Dibutyl
phthalate (CAS No. 84-74-2) were both solved in ethanol, and
ethanol extreme concentrations were both far below 0.5%.
Neodymium(III)nitratehexahydrate(CASNo.16454-60-7)was
dissolvedinH2
O.2NHClwasusedasthesolventforantimony
(III) oxide (Sigma, CAS No. 1309-64-4), and the final HCl con-
tent in medium had no effect on medium pH. Solvent control
was set in every test to deplete the influence of the solvent.
Classification embryotoxicity of the tested compounds
According to the validated prediction model (PM) of the EST
established previously (Genschow et al. 2000; 2002; 2004),
the embryotoxic potential of compounds was classified into
three grades including strongly, weakly, and non-embryo-
toxic. The three endpoints (IC50
3T3, IC50
ES, ID50
) obtained
from differentiation assay and cytotoxic assay were involved
in three linear discriminant functions (I, II, III). The values of
the three functions determined embryotoxic classification.
If IC50
or ID50
concentration exceeded 1000 µg/ml, it was set
to 1000 µg/ml by definition.
Linear discriminant functions I, II, III:•
I: 5.916 l g (IC50
3T3) + 3.500 lg (IC50
ES) − 5.307 [(IC50
3T3-
ID50
)/IC50
3T3] − 15.27
II: 3.651 l g (IC50
3T3) + 2.394 lg (IC50
ES) − 2.033 [(IC50
3T3-
ID50
)/IC50
3T3] − 6.85
III: −0.125 l g (IC50
3T3) − 1.917 lg (IC50
ES) + 1.500
[(IC50
3T3-ID50
)/IC50
3T3] − 2.67
Classification criteria:•
Class 1: Non-embryotoxic, If I > II and I > III
Class 2: Weakly embryotoxic, If II > I and II > III
Class 3: Strongly embryotoxic, If III > I and III > II
Statistics analysis
The statistical analysis was performed using SPSS version
13.0. Data were expressed as mean ± SEM. Each data point
represented three independent experiments. Criterion for
significance was p < 0.05.
Results
Six selected compounds formerly involved in cosmet-
ics, including hydroquinone, eugenol, dibutyl phthalate,
antimony (III) oxide, neodymium (III) nitrate hexahydrate,
and melamine were assessed to show the embryotoxic
potentials by the embryonic stem cell test (EST). 5-Fluor-
ouracil and penicillin G were used as positive and nega-
tive control, respectively. E14 ES cell line or 3T3 cells were
exposed to compounds over a 10 day experiment period.
Three end-points of each compound were analyzed. The
cytotoxicity of the compound on E14 cells and 3T3 cells
(IC50
3T3, IC50
ES) were determined by MTT assay, and
the inhibition of cardiomyocyte differentiation of embry-
onic stem cells (ID50
) was determined by microscopic
observation on day 10.
The values of IC50
3T3, IC50
ES, and ID50
of each compound
were calculated by concentration–response curves through
three independent experiments (Table 1), the sensitivity of
the cytotoxicity assay of ES cells and 3T3 cells, and the dif-
ferentiation assay of ES cells was analyzed. For each com-
pound, concentration–response curves of cytotoxicity assay
and differentiation assay were drafted together (Figure 1).
Three parameters of each compound were substituted into
three linear discriminant functions (I, II , III) in the predic-
tion model (PM), and the classification of embryotoxic
potential of each compound was ascertained (Table 2).
5-Fluorouracil
5-FluorouracilwasusedaspositivecontrolintheEST.5-Fluor-
ouracil displayed intense cytotoxicity to 3T3 cells and ES cells
at very low concentration (IC50
3T3, 0.264 ± 0.0473 µg/ ml,
IC50
ES, 0.062 ± 0.0042 µg/ml), and inhibited the survival
of both cells in a dose-dependent manner (Figure 1A). ES
cells were more sensitive to 5-Fluorouracil than 3T3 cells,
and IC50
ES was much lower than IC50
3T3. 5-Fluorouracil
also possessed strong inhibition for ES cells differentiated
into contracting cardiomyoctyes (ID50
0.050 ± 0.0041 µg/ml)
Table 1. The information of all the test compounds including 5-fluorouracil, penicillin G, hydroquinone, eugenol, dinbutyl phthalate, antimony (III)
oxide, neodymium (III) nitrate hydrate, and melamine.
Product Name CAS# Supplier Formula Alternative name
5-Fluorouracil 51-21-8 Sigma C4
H3
FN2
O2
2,4-Dihydroxy-5-fluoropyrimidine
Penicillin G 69-57-8 Sigma C16
H17
N2
NaO4
S Benzylpenicillin sodium salt
Hydroquinone 123-31-9 Alfa Aesar C6
H6
O2
1,4-Dihydroxybenzene Quinol
Melamine III 108-78-1 Alfa Aesar C3
H6
N6
2,4,6-Triamino-1,3,5-triazine
Eugenol 97-53-0 Alfa Aesar C10
H12
O2
4-Allyl-2-methoxyphenol4-Allylguaiacol
Neodymium (III) nitrate hexahydrate 16454-60-7 Alfa Aesar Nd(NO3
)3
•6H2
O None
Dibutyl phthaiate 84-74-2 Alfa Aesar C16
H22
O4
Dinbutyl phthaiatePhthalic acid di-n-
butyl ester
Antimony (III) oxide 1309-64-4 Sigma Sb2
O3
None
Forpersonaluseonly.

4 Rui Chen et al.
and like the cytotoxicity assay in a dose-dependent manner.
Among the three parameters, ID50
was the lowest, and this
indicated that the differentiation of ES cells was the most
sensitive to 5-fluorouracil. By the prediction model (PM),
5-Fluorouracil was classified with strong embryotoxicity. The
values of IC50
3T3, IC50
ES, and ID50
were in the same range
as the mean values of the ECVAM validation study, and the
embryotoxicity classification was the same as the ECVAM
validation study.
Penicillin G
Penicillin G was non-embryotoxic in the validation study
and set as negative control in the EST. Penicillin G showed
no cytotoxicity to 3T3 cells and ES cells till the concentration
exceeded 1000 µg/ml (Figure 1B). ES cells displayed a little
more sensitivity to Penicillin G than 3T3 cells, and the value
of IC50
3T3 (4043 ± 108.3 µg/ml) was slightly higher than that
of IC50
ES (2076 ± 140.6 µg/ml). Compared with the value of
IC50
3T3 and IC50
ES, The ID50
value (681.1 ± 29.01 µg/ml) was
120%
A
C
E
G
F
H
D
B
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
1000 µg/ml
5 - Fluorouracil
120%
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
COntraction
1000 µg/ml
Hydroquinone
120%
100%
80%
60%
40%
20%
0%
0 10000.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
10000 ng/ml
Antimony (III) oxide
120%
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
1000 µg/ml
Dibutyl phthaiate
120%
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
1000 µg/ml
Melamine
120%
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
1000 µg/ml
Neodymium(III) nitrate hydrate
120%
100%
80%
60%
40%
20%
0%
0 10000.01 0.1 1 10 100
3T3 MTT
ES MTT
Contraction
10000 µg/ml
Pencillin G
120%
100%
80%
60%
40%
20%
0%
0 0.001 0.01 0.1 1 10 100
3T3 MTT
ES MTT
COntraction
1000 µg/ml
Eugenol
Figure 1. Concentration response curves for the compounds: (A) 5-Fluorouracil, (B) penicillin G, (C) hydroquinone, (D) eugenol, (E) dibutyl phthalate,
(F) antimony (III) oxide, (G) neodymium (III) nitrate hexahydrate, and (H) melamine. Each compound contained three concentration response curves
obtained from cytotoxicity assay of 3T3 cells and ES cells and differentiation assay of ES cells, illustrating the effects of the compound on survival of 3T3
cells and ES cells and differentiation of ES cells. (See colour version of this figure online at www.informahealthcare.com/txm)
Forpersonaluseonly.

the lowest, and the differentiation of ES cells showed more
sensitivity than cytotoxicity assay. Penicillin G was classified
as non-embryotoxic, and the classification was in accord
with the ECVAM validation study.
Hydroquinone
3T3 cells and ES cells were both sensitive to hydroqui-
none, the values of IC50
3T3 (5.97 ± 0.485 µg/ml) and IC50
ES
(2.57 ± 0.104 µg/ml) were both below 10 µg/ml (Figure 1C). ES
cells were a little more sensitive than 3T3 cells. Surprisingly,
the effects of hydroquinone on ES cells differentiation were
slightly more intensive than the effects on ES cells viability,
and the value of ID50
(3.77 ± 0.306 µg/ml) was a little higher
than that of IC50
ES (2.57 ± 0.104 µg/ml). Hydroquinone was
classified as strongly embryotoxic.
Eugenol
In the cytotoxicity assay, the viability of 3T3 cells and ES
cells decreased by 50% at very low concentration of eugenol
(Figure 1D). The IC50
3T3 value (3.58 ± 0.209 µg/ml) was
about twice the IC50
ES value (1.85 ± 0.128 µg/ml), but it was
approximated to the ID50
(3.43 ± 0.168 µg/ml). Among three
assays, the cytotoxicity assay of ES cells reached its endpoint
at first, then was the differentiation of ES cells, and the last
was the cytotoxicity assay of 3T3 cells. Eugenol showed its
strong inhibition on the survival of 3T3 cells and ES cells
and the cardiac differentiation of ES cells, it was defined as
a strong embryotoxic compound by PM.
Dibutyl phthalate
Under dibutyl phthalate treatment, greater cytotoxicity
effects were found on ES cells than on 3T3 cells (IC50
3T3
321.2 ± 8.60 µg/ml, IC50
ES 180.6 ± 4.84 µg/ml). ES cells
reached the test end-points at a much lower concentration
in the differentiation assay than in the cytotoxicity assay
(Figure 1E), and the ID50
value (34.4 ± 3.91 µg/ml) was only
~1/5 of the IC50
ES value. Dibutyl phthalate was labeled as a
weak embryotoxic compound.
Antimony (III) oxide
Antimony (III) oxide showed intense cytotoxicity to 3T3
cells and ES cells, the inhibition on the survival of 3T3 cells
and ES cells appeared at very low concentration (IC50
3T3
3.56 ± 0.335 µg/ml, IC50
ES 1.42 ± 0.163 µg/ml). ES cells were
more susceptive than 3T3 cells (Figure 1F). There was lit-
tle difference between the effects of antimony (III) oxide
on the viability and differentiation of ES cells, and the ID50
(1.22 ± 0.139 µg/ml) was much closer to the IC50
ES. The nan-
opowder Antimony (III) oxide was identified as a compound
with strong embryotoxicity.
Melamine
The cytotoxicity assay displayed melamine had much weaker
cytotoxicitythanantimony(III)oxide,sodidthedifferentiation
assay (Figure 1G). ES cells were still more sensitive than 3T3
cells, the IC50
3T3 and IC50
ES were both above 100 µg/ml (IC50
3T3 490.9 ± 40.07 µg/ml, IC50
ES 294.1 ± 22.50 µg/ml). The dif-
ferentiation assay showed that melamine also did not have the
same strong inhibition on ES cells differentiation as antimony
(III) oxide, and the ID50
value was 145.2 ± 18.85 µg/ml. The PM
decided melamine was a weak embryotoxic compound.
Neodymium (III) nitrate hexahydrate
In the MTT assay, neodymium (III) nitrate hexahydrate
showed weak cytotoxicity on 3T3 cells and ES cells (IC50
3T3
541.4 ± 19.14 µg/ml, IC50
ES 374.8 ± 21.83 µg/ml), and ES cells
were more susceptible to the cytotoxic effects of neodymium
(III) nitrate hexahydrate than 3T3 cells (Figure 1H). The dif-
ferentiation of ES cells into contracting cardiomyoctyes was
a little more sensitive than the MTT assay of 3T3 cells (ID50
188.7 ± 15.34 µg/ml). Neodymium (III) nitrate hexahydrate
was concluded to possess weak embryotoxicity.
Discussion
The embryotoxicity refers to the side-effects of a substance
that hazards the development of embryo, and it may lead to
growth retardation, malformation, and death. During the past
30 years, at least 30 alternate in vitro methods were established
to test the embryotoxicity of chemical and physical substances
(Brown et al. 1995; Spielmann 1998). Among these methods,
the EST is the only one that uses established cell lines but not
pregnant animals. In the validation study of the EST by the
ECVAM,20codedtestchemicals,classifiedasstrongly,weakly,
Table 2. Three end-point values of EST and embryotoxicity classifications for 5-Fluorouracil, Penicillin G, and six selected compounds formerly involved
in cosmetics including hydroquinone, eugenol, dinbutyl phthalate, antimony (III) oxide, neodymium (III) nitrate hydrate, and melamine. The values of
IC50
3T3, IC50
ES, and ID50
of each compound were calculated by concentration–response curves through three independent experiments, and data are
presented as IC50
and ID50
values (µg/ml) ± SEM. Embryotoxicity was classified as strongly, weakly, or non-embryotoxic by the prediction model (PM).
5-Fluorouracil, hydroquinone, eugenol, and antimony (III) oxide were classified as strong embryotoxic compounds; dinbutyl phthalate, neodymium
(III) nitrate hydrate, and melamine exhibited as weak embryotoxic compounds; Penicillin G as non-embryotoxic compound.
Tested compound IC50
3T3 IC50
ES ID50
EST classification
5-Fluorouracil 0.264 ± 0.0473 0.062 ± 0.0042 0.050 ± 0.0041 Strong
Penicillin G 4043 ± 108.3 2076 ± 140.6 681.1 ± 29.01 Non
Hydroquinone 5.97 ± 0.485 2.57 ± 0.104 3.77 ± 0.306 Strong
Eugenol 3.58 ± 0.209 1.85 ± 0.128 3.43 ± 0.168 Strong
Dibutyl phthaiate 321.2 ± 8.60 180.6 ± 4.84 34.4 ± 3.91 Weak
Antimony (III) oxide 3.56 ± 0.335 1.42 ± 0.163 1.22 ± 0.139 Strong
Melamine 490.9 ± 40.07 294.1 ± 22.50 145.2 ± 18.85 Weak
Neodymium (III) nitrate hexahydrate 541.4 ± 19.14 374.8 ± 21.83 188.7 ± 15.34 Weak
Forpersonaluseonly.

6 Rui Chen et al.
or non-embryotoxic on the basis of their in vivo effects in ani-
mals and/or humans (Smith et al. 1983) were tested in four
laboratories. The outcome showed that the EST can be con-
sidered to be a scientifically validated test, which is ready for
consideration for use in assessing the embryotoxic potentials
of chemicals for regulatory purposes (Genschow et al. 2004).
In the current study, we aimed at applying the EST in the
cosmetics field. Mouse embryonic stem cell line E14 was used
intheESTasasubstituteforD3celllineintheECVAMESTval-
idationtest.5-FluorouracilandpenicillinGweresetascontrols
to evaluate the reliability of our findings. Our results showed
that 5-Fluorouracil and penicillin G were respectively classi-
fied as strongly and non-embryotoxic, and the results were in
accordancewiththeESTvalidationtest.Itindicatedthatmouse
embryonicstemcelllineE14usedinoursystemscouldreplace
D3celllineusedintheESTvalidationtest withoutaffectingthe
embryotoxic classification of compounds.
In the study, we detected the embryotoxicity of the six
selected tested compounds formerly involved in cosmetics
by the EST. For hydroquinone, it did not show significant
effects on the embryo development in rats and rabbits
(Krasavage et al. 1992; Murphy et al. 1992). Although the
teratogenic potential of hydroquinone to chick embryos
was unremarkable, it displayed embryotoxic at higher
doses (Burgaz et al. 1994). Hydroquinone had also been
found to induce micronuclei transplacently in fetal liver
cells after gastric intubation to pregnant Swiss CD-1 mice
(Ciranni et al. 1988), and it indicated that hydroquinone
had a certain in vivo embryotoxicity in mice. Our in vitro
results revealed hydroquinone with strong embryotoxic-
ity. As to eugenol, it did not have developmental adverse
effects in mice at 100 mg/kg (Amini et al. 2002). For its
isomer-isoeugenol, it led to intrauterine growth retarda-
tion and mildly delayed skeletal ossification in rats at
the dose of 1000 mg/kg (George et al. 2001; NTP 1999).
Eugenol also displayed with genotoxicity and showed a
dose-dependent inhibitory effect on topoisomerase II
activity (Maralhas et al. 2006). Eugenol was confirmed
with strong embryotoxic potential by our data. With regard
to dibutyl phthalate, there were reports indicating dibutyl
phthalate with teratogenic effects in the male reproductive
system after prenatal exposure (Gray et al. 2000; Barlow
and Foster 2003; Swan et al. 2005). In the established EST,
dibutyl phthalate was defined as weakly embryotoxic, the
ID50
was much lower than IC50
, and it was in accordance
with its teratogenic effects. As to antimony, only a few
reports were published on its teratogenicity (Leonard
and Gerber 1996) and Imai and Nakamura (2006) reported
antimony was revealed to be weakly embryotoxic by the
EST, with regard to antimony (III) oxide, the teratogenicity
was unknown. Our results indicated antimony (III) oxide
with strong embryotoxicity. Melamine was well known
for its side-effects to produce stones in bladders and kid-
neys (Heck and Tyl 1985) and its carcinogenesis (Melnick
et al. 1984); however, no reports on its embryotoxicity
were published. According to our results, melamine was
weakly embryotoxic. With regard to neodymium (III)
nitrate hexahydrate, only one report indicated a mixture
of nitric acid compounds of several rare earth elements
(La, Ce, Nd, Pr, and Sm) was not strong embryotoxic in
rats (Ji and Cui 1988), the single effects of neodymium (III)
nitrate hexahydrate wereunknown. In our study, neodym-
ium (III) nitrate hexahydrate was revealed to be weakly
embryotoxic by the results.
Conclusions
In summary, as shown in the study, the classifications of
embryotoxic potentials of six selected compounds formerly
involved in cosmetics by the established EST are mostly in
accordance with the in vivo results reported before. Our
pilot study may provide a valuable attempt to expand the
application of EST to the cosmetics field.
Declaration of interest
This work was supported by the Key Scientific and
Technological Projects of Guangdong Province
(2007A032100003) and the science and technology plan of
Guangdong Province (2007B030702006). The authors report
no conflicts of interest. The authors alone are responsible for
the content and writing of the paper.
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