JOURNAL OF APPLIED TOXICOLOGY

J. Appl. Toxicol. 19, 163–165 (1999)

Comparison of Cytotoxicity of Various

Surfactants Tested on Normal Human
Fibroblast Cultures using the Neutral Red
Test, MTT Assay and LDH Release
B. Arechabala, C. Coiffard, P. Rivalland, L. J. M. Coiffard and Y. de Roeck-Holtzhauer
Laboratory of Cosmetology and Industrial Pharmacy, Rue du Moulin de la Rousselière, 44805 Saint Herblain, France

Key words: cytotoxicity; surfactants; neutral red test; MTT assay; LDH release; fibroblast cultures.

We used the neutral red test, MTT assay and lactate dehydrogenase (LDH) release to compare the
potential cytotoxicity of six surfactants belonging to different classes—three non-ionic surfactants
(Triton ⴛ100, octylphenoxypolyethoxy alcohol, from Orion; Tween 60, polyoxyethylene (20) sorbitan
monostearate, from ICI Speciality Chemicals; Tween 80, polyoxyethylene (20) sorbitan monolaurate,
from Labosi), two anionic surfactants (Texapon K1298, sodium lauryl sulphate, from Henkel; Texapon
N40, sodium laurylether sulphate, from Henkel) and one cationic surfactant (benzethonium chloride,
from Siber Hegner)—on human fibroblast cultures. According to the LC50 (␮g mlⴚ1), the tested
surfactants can be classified in the following order of increasing cytotoxicity: Tween 80 ⬍ Texapon
N40 ⬍ Tween 60 ⬍ Texapon K1298 ⬍ Triton ⴛ100 ⬍ benzethonium chloride. Copyright  1999 John
Wiley & Sons, Ltd.

trypsinized (0.25% trypsin from Eurobio) and resus-

INTRODUCTION pended in RDMI medium.

The current studies have been carried out to examine
whether in vitro cellular techniques can replace animal
experiments in investigating the toxicity of cosmetic
products. This represents a way in which to comply
with the European regulations that will be applied in
the near future.
We assessed the cytotoxicity of six surfactants
belonging to distinct physicochemical classes on nor-
mal human fibroblasts cultures. We determined the
cellular viability, after surfactant exposure, using the
neutral red test, MTT assay and lactate dehydrogenase
(LDH) release.
EXPERIMENTAL
Fibroblast cultures
The cells used were human fibroblasts obtained from
a plastic surgery service and isolated as described
previously.1,2 The culture medium consisted of RPMI
1640 medium (Gibco Europe, Paisley, UK) sup-
plemented with 5% fetal calf serum (FCS), l-glutamine
(0.2 mM) and penicillin G (100 IU ml⫺1). The cultures
were incubated at 37°C in a humidified 95% air/5%
air/5% CO2 atmosphere. The fibroblasts were then
* Correspondence to: Prof de Roeck-Holtzhauer, Laboratory of
Cosmetology and Industrial Pharmacy, Rue du Moulin de la Rousseli-
ère, 44805 Saint Herblain, France.
CCC 0260–437X/99/030163–03$17.50
Copyright  1999 John Wiley & Sons, Ltd.
Treatment with compounds
The fibroblasts used in these experiments, performed
in triplicate, were between the fourth and eleventh
passages. They were seeded on glass slides placed in
square petri dishes. After incubation for 1 h the
medium was removed and the treatment compound
diluted in culture medium was added.
The six commercially available surfactants to be
tested are:
(1) Benzethonium chloride (from Siber Hegner, Niri-
bel, France).
(2) Triton ⫻100 (octylphenoxypolyoxy alcohol, from
Orion, Nantes, France).
(3) Tween 60 (polyoxyethylene (20) sorbitan mono-
laurate, from Labosi, Elancourt Cedex, France).
(4) Tween 80 (polyoxyethylene (20) sorbitan monoste-
arate, from ICI Speciality Chemicals, Nantes,
France).
(5) Texapon K1298 (sodium lauryl sulphate, from
Henkel, Düsseldorf, Germany).
(6) Texapon N40 (sodium laurylether sulphate, from
Henkel, Düsseldorf, Germany).
Determination of the toxic effect
In the last few years, several in vitro systems for
predicting the effect of toxic compounds as an alterna-
tive to animal tests have been described.3–5
Here, the toxicity has been shown using a colori-
metric method with neutral red, the MTT assay and
an enzymatic method using LDH.
Received 22 April 1998
Revised 25 September 1998
Accepted 30 September 1998
164 B. ARECHABALA ET AL.
Neutral red test
The neutral red dye in phosphate buffer diffuses
through the cytoplasmic membrane and then accumu-
lates in the cytoplasm of the living cell.6–10 The
absorbance read at 540 nm is directly proportional to
the number of live cells in the medium. We used a
Titertrek Uniskan II spectrophotometer and the reading
was done against a standard of phosphate buffer. The
toxicity is calculated by reporting the mean optical
density read for the concentration of the toxic com-
pound in relation to that for the standard.
The MTT test
The cytotoxicity of these surfactants was also determ-
ined with MTT assay.11 The MTT test was chosen to
study how to prevent any damage to the fibroblasts.12,13
A solution of MTT (dimethylthiazol diphenyl tetrazol-
ium bromide, from ICN, Orsay, France) at a concen-
tration of 0.5 mg l⫺1 in phosphate buffer (PBS, from
ICN, Orsay, France) was prepared and sterilized by
filtration. A 250 ␮l aliquot of the product solution to
be tested was put in each well. After a given contact
time, the product was eliminated, each well was rinsed
with phosphate buffer and 250 ␮l of MTT solution
was added to each well. After 4 h of exposure, the
supernatant was eliminated and 250 ␮l of dimethyl-
suphoxide (DMSO, from ICN, Orsay, France) was
added to each well in order to solubilize the blue
crystals of formazan that had been formed. The
absorbance of different solutions was then measured
using a Titertrek Uniskan II spectrophotometer at
570 nm.
Lactate dehydrogenase release
The three surfactants benzethonium chloride, sodium
lauryl sulphate and Triton ⫻100,14 each belonging to
a different class, were tested for LDH release.
Leakage of enzymes such as LDH is a well-known
marker of damage to the cellular membrane15
LDH
CH3-CO-COO− J→ CH3-CHOH-COO−
NADH ⫹ H⫹ NAD⫹
The method used here is based on the determination
of NADH disappearance with a spectrophotometer at
the absorbance value of 340 nm. Decrease in the
absorbance at 340 nm is linked directly to the quantity
of LDH released by the cells in the culture environ-
ment. To achieve the cytotoxicity test, the culture
medium, after the surfactant has acted, is poured into
a clean well at 0°C to determine LDH. The medium
was NADH (25 mg ml⫺1; Sigma, Saint Quentin,
France) and sodium pyruvate (1 mg ml⫺1) in phosphate
buffer (phosphate buffer from Dulbecco’s medium
without Mg or Ca; Flow Laboratories, Nantes, France).
The medium was then maintained at 30°C for 6 min.
Reading of the absorbance was made at 340 nm with
a UV–visible, double-beam spectrophotometer (model
U-2000). The surfactant concentrations tested were:
10⫺3, 10⫺2 and 10⫺1 g l⫺1 for Texapon K1298;
10⫺3, 10⫺2, 10⫺1, 1 and 10 g l⫺1 for Triton ⫻100;
and 10⫺4, 10⫺3, 5.10⫺3, 10⫺2, 10⫺1 and 1 g l⫺1 for
benzethonium choride.
Copyright  1999 John Wiley & Sons, Ltd.
Table 1. Cytotoxicity of three surfactants determined with
the neutral red method
Surfactant tested lc50 ⫾ SDa (␮g ml⫺1)
Benzethonium chloride 8.0 ⫾ 0.9
Texapon K1298 62.0 ⫾ 22.0
Texapon N40 290.0 ⫾ 10.0
Triton ⫻100 34.0 ⫾ 2.0
Tween 60 210.0 ⫾ 15.0
Tween 80 850.0 ⫾ 26.0
a
Mean concentration ⫾ standard deviation calculated from five
experiments resulting in 50% inhibition in the neutral red
uptake.
Table 2. Cytotoxicity of three surfactants determined with
the MTT assay
Surfactant tested lc50 ⫾ SDa (␮g ml⫺1)
Benzethonium chloride 3.5 ⫾ 0.9
Triton ⫻100 41.0 ⫾ 5.0
Tween 80 210.0 ⫾ 15.0
a
Mean concentration ⫾ standard deviation calculated from five
experiments resulting in 50% inhibition with the MTT assay.
RESULTS AND DISCUSSION
The results of the neutral red test are given in Table
1. Mean, standard deviation and standard error were
determined and statistical significance was evaluated
with Student’s t-test. These results allow the tested
surfactants to be classified in increasing order of cyto-
toxicity Tween 80 ⬍ Texapon N40 ⬍ Tween 60 ⬍
Texapon K1298 ⬍ Triton ⫻100 ⬍ benzethonium
chloride. In this case, there is a good correlation with
in vivo results during the occular irritation tests. In
those tests, benzethonium chloride is regarded as sever-
ely irritating, Triton ⫻100 and Texapon K1298 slightly
irritating and Tween 80 non-irritating.
The results concerning the MTT assay are given
in Table 2. The classification in increasing order of
cytotoxicity is Tween 80 ⬍ Triton ⫻100 ⬍ benzethon-
ium chloride. There is no change in this classification
compared to the neutral red test. The lc50 values can
be compared except that for Tween 80; however, this
has no influence concerning its own cytotoxicity level.
The LDH assay (Table 3) did not show significant
differences concerning the 10⫺3 and 10⫺2 g l⫺1
Texapon K1298 concentrations but there is a steady
decrease of absorbance for the 10⫺1 g l⫺1 gl⫺1 concen-
tration. According to this concentration, Texapon
K1298 is cytotoxic. These results match those of
Borenfreund,6,7 giving Texapon K1298 a value of
62.0 ␮g ml⫺1 for the RN-90 (RN-90 = LC90 value with
the neutral red method). This value corresponds to a
cytotoxicity of 10% obtained with the neutral red test.
Thus, for a lower concentration of 10⫺2 g l⫺1, for
example, there will be no toxicity observed, but for a
higher concentration of 10⫺1 g l⫺1, for example, the
cytotoxicity will be more than 10%. For Triton ⫻100,
J. Appl. Toxicol. 19, 163–165 (1999)
 CYTOTOXICITY OF SURFACTANTS ON HUMAN FIBROBLASTS 165

there is no decrease of absorbance for a concentration

of 10⫺3 g l⫺1. A slight decrease occurs when 10⫺2 g l⫺1 Table 3. Decrease of absorbance at 340 nm with the
LDH method
is reached and this is confirmed up to 1 and 10 g l⫺1.
The conclusion is that the concentration for which Substance Concentration Absorbance ⌬A
Triton ⫻100 begins to be cytotoxic is between 10⫺2 tested (g l−1) A
and 10⫺1 g l⫺1. The RN-90 value proposed by Boren-
freund for Triton ⫻100 is 10 ␮g l⫺1. For benzethonium NADPH 2.5 mg ml⫺1 0.756 –
chloride at concentrations of 10⫺4 and 10⫺3 g l⫺1 no
significant decrease of absorbance is noticed but a Texapon K1298 10⫺3 0.750 0.006
slight diminution is noticed at 5 ⫻ 10⫺3 g l⫺1. This 10⫺2 0.745 0.011
absorbance decrease at 340 nm is confirmed by the 10⫺1 0.716 0.040
10⫺2 g l⫺1 concentration and decreases further with the 1 0.701 0.051
other concentrations tested. The results agree with the Triton ⫻100 10⫺3 0.741 0.015
in vivo toxicity of benzethonium chloride, which is 10⫺2 0.732 0.024
considered to be highly irritating to the eye. Boren- 10⫺1 0.711 0.045
freund gives it an RN-90 value of 5 ␮g ml⫺1. When 1 0.700 0.056
comparing the three results, benzethonium chloride is
the most toxic tested by this method. If the 10⫺2 g l⫺1 Benzethonium chloride 10⫺4 0.752 0.004
concentration is considered to be discriminatory, then 10⫺3 0.743 0.013
Texapon K1298 and Triton ⫻100 are non- or slightly 5 ⫻ 10⫺3 0.726 0.020
toxic and benzethonium chloride is highly toxic. 10⫺2 0.724 0.032
The results given by this last method correlate well 10⫺1 0.705 0.051
1 0.695 0.161
with the other two tests. It would be interesting to
design tests with the aim of determining the decrease
of absorbance at intervals of 1 min.
These cytoxicity tests on human fibroblast cultures
of different surfactants belonging to different classes
allowed a comparison of effects with other methods
and proved useful for classifying cosmetics.

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Copyright  1999 John Wiley & Sons, Ltd. J. Appl. Toxicol. 19, 163–165 (1999)