Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

Article abstract of DOI:10.1016/j.mrgentox.2003.09.004

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01-0.5mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.

Full text of DOI:10.1016/j.mrgentox.2003.09.004

Mutation Research 542 (2003) 117–128
Induction of chromosome aberrations in cultured human
lymphocytes treated with ethoxyquin
A. Błaszczyka,∗, R. Osieckaa, J. Skolimowskib
Department of Cytogenetics and Plant Molecular Biology, University of Łódz´, Banacha 12/16, 90-237 Łódz´, Poland
a
b
Department of Organic Chemistry, University of Łódz´, Narutowicza 68, 90-136 Łódz´, Poland
Received 5 March 2003; received in revised form 8 August 2003; accepted 12 September 2003
Abstract
The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preserva-
tive, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken
because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing
out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incu-
bated with EQ (0.01–0.5 mM) both with and without metabolic activation. Stability studies performed by HPLC analysis
showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration
assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation
chromosomes.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Ethoxyquin; Food preservative; Chromosome aberrations; Human lymphocytes
1. Introduction
itself was not mutagenic to TA 88 and TA 100 S.
typhimurium strains but it enhanced the mutagenic
activity of DMAB (3,2ꢀ-dimethyl-4-aminobiphenyl).
On the other hand, anti-mutagenic EQ effects were
observed both in the Ames test and in in vivo tests
on animals (mice, rats, and Chinese hamsters) [7–9].
It was shown that EQ reduced the number of chro-
mosome aberrations, micronuclei, and dominant
lethal mutations induced by the alkylating agent cy-
clophosphamide, a mutagen acting after metabolic
activation. In contrast, the formation of sister chro-
matid exchanges (SCE) induced by this mutagen was
not influenced by EQ, probably because of differ-
ent mechanism for these events. The above studies
also showed that EQ by itself was not mutagenic in
the performed in vivo tests [7,8]. Burka et al. [10]
studied the metabolism of EQ in rats and mice, and
Ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-tri-
methylquinoline) is an antioxidant compound widely
added to animal food. It is also used as a color
preservative in paprika, chili powder, and ground
chili as well as an anti-degradation agent used in
rubber production. The mutagenicity of EQ has been
tested on prokaryotic organisms, mainly strains of
Salmonella typhimurium (Ames test). The results
obtained were equivocal as some authors [1–3] re-
ported negative results while others observed positive
responses [4,5]. Reddy et al. [6] observed that EQ
∗
Corresponding author: Tel.: +48-42-635-44-26;
fax: +48-42-635-44-23.
E-mail address: ablasz@biol.uni.lodz.pl (A. Błaszczyk).
1383-5718/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2003.09.004

118
A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
identified several metabolites. It was shown that EQ
was extensively metabolized, but other authors [11]
reported the presence of the parent compound in rat
bile after administration of EQ to the tested animals.
These different results could have been caused by
differences between strains of rats used [10]. Burka
et al. [10] also showed that the major products of EQ
metabolism were not the same in rats and mice: mice
produced more glucoronide metabolites, whereas
in rats more sulfate metabolites were observed. It
means that the EQ metabolic pathways can be differ-
ent.
Since there are no published data on the mutagenic
activity of ethoxyquin in animal and human cells in
vitro, in the present study EQ genotoxicity was as-
sessed in the chromosomal aberration test on cultured
human lymphocytes. EQ mutagenic properties were
tested without metabolic activation, and also after ap-
plication of S9 fraction induced in rats by Aroclor
1254. In the report on ethoxyquin, the results of exper-
iments concerning environmental exposure to EQ and
its stability performed by Monsanto Industrial Chem-
icals Company have been presented [12]; the results
obtained suggested that EQ might be chemically un-
stable. It is also known that EQ can spontaneously
convert into different oxidation products (which are
also effective antioxidants) [13]. For this reason we
also studied, using the HPLC method, the stability of
EQ in cell culture and the degree of its metabolization
by the S9 fraction.
vacuum and the residue distilled. EQ was separated
as a light yellow liquid by the use of Kugel–rohr
redistillation.
NMR : 13C NMR (CDCl3) 14.9 (2-CH3);
18.4 (2-CH3); 30.1 (4-CH3); 51.5 (C);
64.0 (CH2–O); 110.8; 113.5; 114.2; 122.7;
128.4; 129.5; 137.3 (C; C-5ꢀ) 151.0 (C; C-8ꢀ).
2.1.2. Preliminary studies
EQ was dissolved in ethanol (ET), and freshly
prepared solutions were added to cultures at appro-
priate final concentrations. The final concentration
of ethanol in all cultures was 0.1% (v/v). In prelim-
inary studies the cytotoxicity of EQ was analyzed.
It was shown that 1.0 mM concentration of EQ was
toxic for human lymphocytes. The highest dose
used in subsequent experiments was 0.5 mM (the
mitotic index (MI) was considerably suppressed),
and the lowest dose was 0.01 mM (no toxicity was
observed).
2.1.3. Positive control
Two positive controls were performed to confirm the
responsiveness of cells to known chromosome aberra-
tion inducers:
• the control with mechlorethamine (ME, Sigma), a
direct-acting clastogen, which was used at the con-
centration of 0.0001 mM;
• the control with cyclophosphamide (CP, Sigma),
an indirect-acting clastogen, which was used at the
concentration of 0.02 mM.
2. Materials and methods
These mutagens were freshly prepared before each
experiment in sterile water (Sigma).
2.1. Chemicals tested
2.1.1. Synthesis of ethoxyquin
The ethoxyquin tested (CAS number: 91-53-2) was
supplied by the Department of Organic Chemistry,
2.2. Stability studies
´
University of Łódz.
The stability of EQ in lymphocyte cultures with-
out and with S9 mix was analyzed by use of
high-performance liquid chromatography (HPLC) ac-
cording to the procedure described by Luckas [14].
The conditions applied were the same as those used
in a chromosome aberration assay performed in geno-
toxicity studies and described below. Samples were
removed from cultures after 2 or 3 h following the
addition of EQ, analyzed after centrifugation of cells,
EQ was obtained from 0.5 mol p-phenetidine
(4-ethoxyaniline) and 0.6 mol diacetone alcohol
(4-hydroxy-4-methyl-2-pentanone), 100 mg p-tolue-
nosulfonic acid by heating in 200 ml toluene with the
use of a Dean–Starks trap during 5 h. The mixture
was heated at 130 ◦C (oil bath) for 3 h and toluene
was distilled from it. The solvent and excess mesity-
loxide (4-methyl-3-penten-2-one) were removed in

A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
119
and compared with initial measurements taken at the
time of treatment. The Knauer liquid chromatograph
consisting of a Model 64 pump, a 250 mm × 4.6 mm
i.d. chromatographic column, Nucleosil 100-7C18, a
variable-wavelength spectrophotometric detector and
a 20 ␮l injection valve was used. Peak areas were
determined by means of a Hewlett–Packard Model
3395 integrator. Chromatographic separation was car-
ried out isocratically at room temperature with an
eluent containing a 0.4% aqueous solution of sodium
formate (pH 7.5) and methanol (40:60, v/v), at a flow
rate 1 ml/min. The mobile phase, prepared by mixing
measured volumes of the solvents, was ultrasonically
degassed before use. The spectrophotometric detector
was set at 246 nm. The EQ retention time in chro-
matographic conditions applied was 12.5 min. The
content of EQ in the samples was qualified from a
calibration curve generated with EQ standards. Peak
areas evaluated by the integrator from the appropriate
chromatograms were plotted as a function of the EQ
concentration.
The S9 mix solution was prepared immediately
before use by mixing 0.8 ml of S9 (prepared from
livers of Aroclor 1254-treated rats) with 0.2 ml 0.2 M
glucose-6-phosphate, 0.2 ml 0.2 M NADP, 0.2 ml
1.65 M KCl, 0.2 ml 0.4 M MgCl2, 5 ml 0.2 M phos-
phate buffer (pH 7.4) and 3.15 ml distilled water (each
from Sigma). The final concentration of S9 used was
1% (v/v).
Colcemid (0.1 ␮g/ml, Sigma) was added to each
culture 2 h before harvesting.
2.4. Chromosome preparation
The cells were harvested by centrifugation
(100 × g, 10 min), given a hypotonic shock in
0.075 M KCl (37 ◦C) for 15 min, and then fixed
three times in a fresh solution of methanol/glacial
acetic acid (3:1, v/v) [15]. Slides were prepared
using usual air-drying technique, stained with 5%
Giemsa (Sigma) and analyzed for chromosome aber-
rations.
2.3. Cell cultures and chemical treatment
2.5. Cytogenetic analysis
Human peripheral blood lymphocytes were ob-
tained from three healthy female donors who had not
been exposed to any known mutagens for 6 months
prior to the cytogenetic investigation.
Blood samples were cultured and then harvested
by standard procedures [15]. The final culture volume
was always 3 ml. Heparinized whole blood (0.3 ml)
was incubated for 72 h in RPMI 1640 medium (Sigma)
supplemented with 15% foetal calf serum (Sigma)
and 1% penicillin/streptomycin solution (Sigma).
Cells were stimulated with 1% phytohemagglutinin
M (Difco).
For each donor, 150 Giemsa-stained metaphases for
each EQ concentration were analyzed (75 metaphases
from single cell culture). At some EQ doses for which
the quality of metaphases was poor or cytotoxicity
was observed, the numbers of analyzed cells were
lower. Only well spread, good quality metaphases with
46 1 chromosomes were analyzed. The percentage
of cells with aberrations, quantity of chromatid- and
chromosome-type breaks and exchanges, total number
of structural aberrations per 100 cells and the number
of polyploid metaphases were scored. Gaps were also
scored but they were grouped separately. Identification
of the aberration type was performed according to the
International System for Human Cytogenetic Nomen-
clature [16].
The genotoxic effect of EQ was analyzed by com-
paring the number of abnormal cells and the chromo-
some aberrations per cell for the different treatments
with the respective control values. The mitotic in-
dex was also determined by scoring 1000 cells from
each donor. Statistical analysis was performed us-
ing the χ2-test and the Student’s t-test. Correlation
co-efficients were calculated to analyze dose-response
relationships.
The EQ was added to cultures according to the three
alternative protocols:
• the lymphocytes were treated for 3 h with EQ 48 h
after initiation of the cultures;
• the lymphocytes were treated with EQ (or with ME
as a positive control) continuously for the last 24 h
of the cultures;
• the lymphocytes were treated for 2 h with EQ (or
with CP as a positive control) in the presence of S9
mix, 48 h after initiation of the cultures.
Duplicate cultures were used for all treatments.

120
A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
3. Results
Tables 1–3 show the results of the chromosome
aberration test obtained for each donor separately.
Analysis of the MI after 3-h treatment of lympho-
cytes with EQ (without metabolic activation, Table 1)
showed that only the higher doses of EQ significantly
suppressed divisions of the treated lymphocytes.
The strongest cytotoxic effect was produced by EQ
at 0.5 mM concentration. The reduction of MI was
about 50% in two donors (number 2 and 3); in cul-
tures of lymphocytes from donor 1 after treatment
with 0.5 mM of EQ no metaphases were observed.
Analysis of chromosome aberrations showed that EQ
induced aberrations mainly of chromatid type (gaps
and breaks); in each of donor atypical translocation
chromosomes were observed (Table 1). Increases in
the number of aberrations in comparison with the
negative controls were observed, but they were not
very high (statistically insignificant). The induction
of polyploid cells by EQ was also seen for two of the
three donors (Table 1).
Results of the HPLC assay showed that EQ was a
very stable compound in lymphocyte cultures in vitro.
The concentrations of EQ observed after its 3 h incu-
bation (without S9 mix) with the cells were very sim-
ilar to the initial ones (Fig. 1). Therefore, EQ was not
degraded and was present and bioavailable under the
same cell culture conditions as used in the chromo-
some aberrations assay.
Analysis of the samples from cultures in which EQ
was added together with S9 mix showed that this com-
pound was effectively metabolized in vitro by enzymes
of the rat liver microsomal fraction (Fig. 1). After a
2 h incubation of EQ with S9 mix in lymphocyte cul-
tures, the concentrations of EQ were much lower in
comparison with the concentrations of the test com-
pound in respective cultures without S9 mix. The con-
centrations of EQ were declining to a different extent
depending on initial EQ concentrations: the percent-
ages of EQ remaining in cultures with 0.5, 0.25, and
0.1 mM concentrations of this compound were about
95, 60, and 26, respectively. In cultures in which the
initial EQ doses were 0.05, 0.025, and 0.01 mM, the
compound was metabolized completely.
Table 2 presents the results of a 24-h treatment
of lymphocytes with EQ. The cytotoxic properties of
EQ were also seen: the MI was significantly reduced
(by over 50%) by EQ at 0.25 mM, while the highest
EQ dose (0.5 mM) inhibited metaphases completely
0,6
0,5
0,4
0,3
0,2
0,1
0
Control
2 h
2 h + S9
0
0,1
0,2
0,3
0,4
0,5
0,6
Concentration of EQ (mM)
Fig. 1. Measured concentration of EQ in lymphocyte cultures after a 2-h treatment with the test compound in the presence of S9 mix, and
after a 3-h treatment without metabolic activation.

Table 1
Chromosome aberrations induced by EQ (3-h treatment) in cultured human lymphocytes in the absence of S9 mix
Donor
no.
EQ dose
(mM)
No. of cells
scored
Mitotic
index (%)
Abnormal cells (%)
No. of structural chromosome
aberrations
No. of structural
aberration per cell
Polploidy
Inclusive
gaps
Exclusive
gaps
A
B
C
D
E
F
G
Total
1
1
1
1
1
1
1
1
0
ET
150
150
150
150
150
150
150
n.m.a
8.1
7.8
9.1
8.0
8.8
4.9∗
6.1
2.0
2.0
2.0
4.0
3.3
3.3
6.0
2.0
2.0
1.3
2.0
3.3
3.3
3.3
1
2
0
2
0
1
3
2
1
1
0
3
2
5
0
0
0
0
1
0
0
0
0
1
1
0
0
1
0
0
1
3
0
3
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
3
3
3
6
5
6
9
0.020
0.020
0.020
0.040
0.033
0.040
0.060
0
0
1
2
4
3
5
0.01
0.025
0.05
0.1
0.25
0.5
2
2
2
2
2
2
2
2
0
ET
150
150
150
150
150
150
150
130
7.6
6.4
6.0
7.8
5.7
5.2
6.0
3.2∗
0.7
1.3
4.0
2.0
2.0
1.3
3.3
2.3
0.0
0.7
3.3
0.7
2.0
0.7
2.7
0.8
1
1
1
1
0
1
1
2
0
1
3
0
2
1
2
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
2
6
3
3
2
5
3
0.007
0.013
0.040
0.020
0.020
0.013
0.033
0.023
0
0
2
5
3
3
3
3
0.01
0.025
0.05
0.1
0.25
0.5
3
3
3
3
3
3
3
3
0
ET
150
150
150
150
150
150
150
140
6.5
6.2
6.1
5.8
5.5
4.9
5.1
3.4∗
1.3
1.3
2.0
2.7
3.3
4.0
3.3
2.1
0.7
0.7
1.3
2.0
2.0
2.7
2.7
1.4
1
1
1
1
2
2
1
1
1
1
2
2
2
2
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
2
2
3
4
5
6
5
3
0.013
0.013
0.020
0.027
0.033
0.040
0.033
0.021
0
0
0
0
0
0
0
0
0.01
0.025
0.05
0.1
0.25
0.5
A: chromatid gap; B: chromatid break; C: chromatid exchange; D: chromosome gap; E: chromosome break; F: dicentric; G: atypical translocation chromosome; H: polyploidy;
ET: ethanol.
an.m.—no metaphase chromosomes were observed.
∗Significantly different from the respective control (P < 0.05).

Table 2
Chromosome aberrations induced by EQ (24-h treatment) in cultured human lymphocytes in the absence of S9 mix
Donor
no.
EQ dose
(mM)
No. of cells
scored
Mitotic
index (%)
Abnormal cells (%)
No. of structural chromosome
aberrations
No. of structural
aberration per cell
Polyploidy
Inclusive
gaps
Exclusive
gaps
A
B
C
D
E
F
G
Total
1
1
1
1
1
1
1
1
1
0
ET
150
150
150
150
150
150
135
n.m.a
150
7.1
7.3
6.7
6.8
6.2
6.0
3.0∗
0.0
0.0
0.7
2.0
2.0
1.3
5.2∗
0.0
0.0
0.7
1.3
0.7
0.7
3.7∗
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
3
2
7
0
0
0
0
1
1
1
1
4
0
0
0
1
1
1
0
1
2
1
1
2
0.01
0.025
0.05
0.1
0.25
0.5
0.007
0.020
0.020
0.013
0.052∗
ME
6.8
22.7∗
20.0∗
8
17
2
1
8
0
0
36
0.240∗
1
2
2
2
2
2
2
2
2
2
0
ET
150
150
150
150
150
150
0b
12.0
11.6
10.0
11.5
11.5
9.2
1.0
1.3
2.7
0.7
3.3
4.7
0.0
0.7
2.7
0.7
2.7
4.0
1
1
0
0
1
1
0
1
3
1
2
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
0
0
1
0
0
0
1
2
4
1
5
0.010
0.013
0.026
0.007
0.033
0.067∗
4
1
3
2
3
1
0.01
0.025
0.05
0.1
0.25
0.5
10
4.5∗
n.m.a
150
ME
11.5
20.0∗
12.7∗
10
10
2
3
7
0
0
32
0.427∗
0
3
3
3
3
3
3
3
3
3
0
ET
150
150
150
150
150
150
0b
5.9
5.8
5.3
6.1
5.0
6.0
2.9∗
2.0
2.0
2.7
4.7
6.7∗
7.3∗
0.7
1.3
2.0
2.0
6.0∗
6.0∗
2
1
1
4
1
2
1
2
2
3
7
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
6
0
0
0
0
0
0
0
0
1
0
0
0
3
3
4
7
13
14
0.020
0.020
0.030
0.047
0.087∗
0.093∗
0
0
1
1
1
1
0.01
0.025
0.05
0.1
0.25
0.5
n.m.a
150
ME
6.8
28.7∗
16.0∗
13
10
6
6
8
0
0
43
0.287∗
1
A: chromatid gap; B: chromatid break; C: chromatid exchange; D: chromosome gap; E: chromosome break; F: dicentric; G: atypical translocation chromosome; H: polyploidy;
ET: ethanol; ME: mechlorethamine (0.0001 mM, positive control).
an.m.—no metaphase chromosomes were observed.
bMetaphases were not acceptable quality.
∗Significantly different from the respective control (P < 0.05).

Table 3
Chromosome aberrations induced by EQ in cultured human lymphocytes in the presence of S9 mix
Donor
no.
EQ dose
(mM)
No. of cells
scored
Mitotic
index (%)
Abnormal cells (%)
No. of structural chromosome
aberrations
No. of structural
aberration per cell
Polyploidy
Inclusive
gaps
Exclusive
gaps
A
B
C
D
E
F
G
Total
1
1
1
1
1
1
1
1
1
1
0
S9
150
150
150
150
150
40
150
150
150
150
7.0
4.6
4.8
4.5
4.2
3.0
6.0
3.0
2.8∗
5.5
1.3
1.3
2.7
2.7
8.7∗
7.5
1.3
1.3
2.0
2.7
4.7
5.0
4.7
4.0
1
1
1
0
4
1
0
7
6
2
1
1
3
4
3
2
3
5
6
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
3
1
2
0
0
0
0
3
0
2
0
1
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
2
2
4
4
13
3
0.013
0.013
0.027
0.027
0.087∗
0.075
0.047
0.107∗
0.100∗
0.387∗
0
0
0
2
1
0
1
1
1
0
ET + S9
0.01
0.025
0.05
0.1
0.25
0.5
CP
4.7
7
10.7∗
10.0∗
33.3∗
16
15
58
5.3
30.7∗
33
10
2
2
2
2
2
2
2
2
2
2
0
S9
150
150
150
150
150
150
150
150
150
150
7.7
8.8
7.3
6.8
6.2
8.2
7.7
6.3
3.9∗
8.9
0.7
1.3
1.3
3.3
2.0
1.3
1.3
4.0
15.3∗
31.3∗
0.7
0.7
1.3
3.3
2.0
1.3
1.3
2.0
13.3∗
28.0∗
0
1
0
1
0
0
0
3
3
6
1
1
2
2
2
2
2
3
14
42
0
0
0
0
0
0
0
0
0
7
0
0
0
0
0
0
0
0
0
3
0
0
0
1
0
0
0
0
4
1
0
0
0
3
0
0
0
0
1
0
0
0
0
0
1
0
0
0
1
0
1
2
2
7
3
2
2
6
23
59
0.007
0.013
0.013
0.047
0.020
0.013
0.013
0.040
0.153∗
0.393∗
0
0
2
0
2
1
2
1
0
0
ET + S9
0.01
0.025
0.05
0.1
0.25
0.5
CP
3
3
3
3
3
3
3
3
3
3
0
S9
150
150
150
150
150
150
150
120
0a
4.5
3.6
3.2
3.0
4.0
3.3
4.0
2.8
3.1
4.6
2.0
2.0
2.0
0
2
1
1
0
8
7
2
4
0
2
2
3
6
4
3
10
0
0
0
0
0
1
0
0
0
0
0
0
2
0
0
2
0
0
0
1
2
3
0
3
0
0
0
0
0
0
0
0
0
0
0
0
2
1
0
1
2
3
3
0.020
0.020
0.020
0.020
0.133∗
0.107∗
0.033
0.167∗
0
0
0
1
2
1
0
0
1.3
1.3
2.0
5.3
5.3
2.0
7.5∗
ET + S9
0.01
0.025
0.05
0.1
0.25
0.5
CP
2.0
3
10.0∗
10.7∗
3.3
20
16
5
12.5∗
20
150
34.7∗
32.7∗
1
30
12
2
9
0
0
54
0.360∗
A: chromatid gap; B: chromatid break; C: chromatid exchange; D: chromosome gap; E: chromosome break; F: dicentric; G: atypical translocation chromosome; H: polyploidy;
S9: the control with S9 mix; ET + S9: the control with the solvent (ethanol) and S9 mix, CP: cyclophosphamide (0.02 mM, positive control).
aMetaphases were not acceptable quality.
∗Significantly different from the respective control (P < 0.05).

124
A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
in samples from all donors. The chromatid breaks
and gaps were the dominant aberrations, but also
dicentrics and atypical translocation chromosomes
were observed (Table 2). The number of aberrations
after EQ treatment increased in all three donors in
comparison with the negative controls. Polyploidy
was observed also for each donor, however, it was
also seen in the negative control of one of them
(Table 2).
Table 3 presents the results after metabolic activa-
tion by the S9 mix. The analysis of MI showed that
EQ was less cytotoxic for human lymphocytes if it
was added to cultures together with S9 mix. For all
donors the reduction of MI values was also seen at
the highest doses of EQ (especially at 0.5 mM) but
it was always below 50%. A cytotoxic effect of S9
alone was also seen (donors 1 and 3). The number
of aberrant cells and aberrations observed was larger
than without metabolic activation. The most frequent
were chromatid breaks and gaps; dicentric and a typi-
cal translocated chromosomes were also observed, as
well as one chromatid exchange.
Fig. 2 shows the pooled results (from three donors)
of the mitotic index and of the chromosome aberra-
tions analysis. EQ at the 0.5 mM concentration sig-
nificantly reduced the mitotic index in each variant
of experiment (Fig. 2A). Fig. 3 shows that the nega-
tive dose–response relationship was rather weak (r =
−0.35) in experiments with metabolic activation of
EQ (Fig. 3C) but the relationship was clearly observed
in experiments without metabolic activation (Fig. 3A
and B).
The numbers of aberrations were significantly in-
creased, especially in experiments with metabolic
activation of EQ and, to a smaller degree, in ex-
periments with a 24-h treatment (Fig. 2B), but they
were significantly lower in comparison with respec-
tive positive controls. Analysis of the dose–response
9
8
7
6
5
4
3
2
1
0
(A)
*
*
*
*
*
NC
0.01
0.025
0.05
0.1
0.25
0.5
PC
EQ concentration (mM)
0,5
0,4
0,3
0,2
0,1
0
(B)
*
*
*
*
*
*
*
*
*
*
NC
0.01
0.025
0.05
0.1
0.25
0.5
PC
EQ concentration (mM)
Fig. 2. The pooled results of the mitotic index (A) and of the chromosome aberration (B) analysis obtained after incubation of human
lymphocytes with EQ (white columns: 3-h treatment, gray columns: 24-h treatment, and black columns: the treatment with S9 mix).
NC, the respective negative controls (ET or ET + S9); PC, positive controls: mechlorethamine (gray) and cyclophosphamide (black). (∗)
Significantly different from the respective negative control (P < 0.05).

A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
125
(A)
(C)
(B)
20
15
10
5
20
15
10
5
20
15
10
5
r = - 0.75
r = - 0.35
r = - 0.59
0
0
0
0
0.1 0.2 0.3 0.4 0.5
0
0.1 0.2 0.3 0.4 0.5
0
0.1 0.2 0.3 0.4 0.5
Concentration (mM)
Fig. 3. Correlation coefficients and regression lines of mitotic index in cultures of human lymphocytes treated with ethoxyquin for (A)
3 h, (B) 24 h, and (C) 2 h in the presence of S9 mix.
(C)
(B)
0,5
0,4
0,3
0,2
0,1
0
0,5
0,4
0,3
0,2
0,1
0
(A)
0,5
0,4
0,3
0,2
0,1
0
r = 0.57
r = 0,47
r = 0,14
0
0,1 0,2 0,3 0,4 0,5
0
0,1 0,2 0,3 0,4 0,5
0
0,1 0,2 0,3 0,4 0,5
Concentration (mM)
Fig. 4. Correlation coefficients and regression lines of chromosome aberrations (including gaps) in cultured human lymphocytes treated
with ethoxyquin for (A) 3 h, (B) 24 h, and (C) 2 h in the presence of S9 mix.
relationship shows that no increase in total chromo-
some aberrations was observed (r = 0.14) together
with the increasing dose of EQ after 3-h treatment
of lymphocytes (Fig. 4A). The number of aberrations
observed after 24 h incubation of EQ with lympho-
cytes increased together with increasing EQ concen-
tration, but the positive dose–response relationship
was not very strong (r = 0.47, Fig. 4B). In the ex-
periments with metabolic activation it was slightly
stronger (r = 0.57, Fig. 4C). Similar relationships
were observed when the number of aberrant cells was
analyzed (Fig. 5).
(C)
20
(A)
(B)
20
15
10
5
20
15
10
5
r = 0,65
15
r = 0,16
r = 0,52
10
5
0
0
0
0
0,1 0,2 0,3 0,4 0,5
0
0,1 0,2 0,3 0,4 0,5
0
0,1 0,2 0,3 0,4 0,5
Concentration (mM)
Fig. 5. Correlation coefficients and regression lines of aberrant cells (including gaps) in cultures of human lymphocytes treated with
ethoxyquin for (A) 3 h, (B) 24 h, and (C) 2 h in the presence of S9 mix.

126
A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
4. Discussion
was not correlated with the dose of the compound
tested. It is possible that the number of aberrations,
like translocated chromosomes, was underestimated.
Translocated chromosomes are not always detected
in Giemsa-stained metaphases; they appear only if
the derived chromosomes are atypical. The use of
other methods of analysis, including the chromosome
painting method (fluorescence in situ hybridization
(FISH)), in future studies on EQ would give more
information on the real number of translocations in-
duced and mechanisms involved in the process of
aberration induction caused by this compound.
EQ induced many gaps; their number was the high-
est in experiments with S9 mix. Scoring and analyzing
gaps in the chromosome aberration test is controver-
sial and the significance of gaps is unknown [23,24]
but they are usually scored and grouped separately.
Recently, Paz-y-Mino et al. [25] evaluated DNA dam-
age in human lymphocytes due to occupational expo-
sure to low levels of ionizing radiation. The authors
used two assays: the comet assay and the chromosome
aberration test. They observed a higher correlation be-
tween the results from these two methods when gaps
were included in the correlation analysis (r = 0.78
versus r = 0.50). The authors concluded that gaps are
indicators of damage in DNA and should be scored in
the chromosome aberration analysis.
After EQ addition to lymphocyte cultures some
polyploid cells were also observed. The assessment
of polyploidy in chromosomal aberration analysis in
vitro can be used as information for the assessment
of the aneugenic potential of a compound, since the
cellular target structures for both phenomena are be-
lieved to be largely identical [26]. It was shown that
the majority of definite aneugens induce polyploidy
in vitro.
The present results indicate that activity of EQ in
inducing different types of chromosome aberrations
was the highest when EQ was metabolically activated
with S9 mix. The experiments performed with HPLC
showed that EQ was metabolized with enzymes of
rat microsomes, but the metabolites were not studied.
The EQ doses: 0.01, 0.025, and 0.05 mM were entirely
metabolized, but in cultures with higher doses of the
tested compound unchanged EQ was present after a
2 h incubation with S9 mix. Thus, at the higher con-
centrations of EQ the cultured lymphocytes were ex-
posed not only to EQ metabolites but to unchanged EQ
The present study was initiated to find out whether
ethoxyquin, a food preservative, is able to induce chro-
mosome aberrations in human lymphocytes in vitro.
Increasing use and high levels of EQ in animal food
justify examination of its genotoxicity. The results ob-
tained in the Ames test have been both positive [1–3,6]
and negative [4,5]; no published in vitro data have been
available on EQ genotoxicity in mammalian cells.
There are reports showing that in animals EQ can
modify carcinogenic response to different carcino-
gens [17,18], and there is also a report indicating
that EQ causes tumors. Manson et al. [19] showed
that EQ completely prevented the formation of afla-
toxin B1-induced preneoplastic lesions in rat liver,
but EQ alone caused severe damage to the kidney.
The observed changes indicated that EQ accelerated
the aging process and that it might be exerting a
carcinogenic effect in the kidney.
The present results show that EQ induced chro-
mosome aberrations in lymphocyte cultures; the
number of aberrations observed was higher as com-
pared with the negative control. The highest num-
bers of aberrations and aberrant cells were observed
after a 24 h incubation of EQ with cells and in ex-
periments using added metabolic activation. The
number of cells with aberrations and the number
of aberrations were not very high compared with
mechlorethamine or cyclophosphamide as positive
controls, but dose–response relationships were ob-
served. The chromatid-type aberrations (breaks, gaps)
were the most frequent at all variants of our exper-
iments. These results suggest that in human lym-
phocyte cultures, EQ can be considered to be an
S-independent agent. It is assumed that chromatid
breaks are the result of DNA double-strand breaks
[20] and it is also well established that such breaks can
be the primary DNA lesions leading to the formation
of chromosomal rearrangements [21]. In our experi-
ments, EQ induced dicentric and translocated atypical
chromosomes as well. These types of aberrations are
known to have serious biological consequences [22].
Dicentric and atypical chromosomes were observed
in each of the three experiments, and also in each
of the donors, but were most numerous in experi-
ments using metabolic activation (S9 mix) of EQ. The
number of these aberrations was not very high and

A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
127
as well. It was shown that EQ induced a wide range
of enzymes (for example GSH transferases, epoxide
hydrolase, cytochrome P450) and all of these enzyme
systems were involved in EQ metabolism [10]. Burka
et al. [10] studied the metabolism of EQ in F344 rats
and B6C3F1 mice and showed that the major path-
ways of EQ metabolism were O-deethylation and con-
jugation to endogenous substrates. Eight metabolites
were detected in rat urine (two major ones in the form
of sulfate conjugates) and no parent EQ was reported.
Mouse urine contained also two major glucuronide and
sulfate conjugates. The differences in metabolites ob-
served in rat and mouse urine indicate that metabolism
of EQ differed significantly between these animals.
In rat bile, three metabolites were found mainly
as GSH conjugates with little unchanged EQ present
(about 5%). Biliary metabolites were also subject of
the study by Skaare [11] who reported that 75–85%
of the radiolabelled material excreted in albino rat
bile following administration of EQ was the parent
compound. The discrepancy between the above re-
sults can be accounted for by assuming a different EQ
metabolism in the rat strains used, but it is also possi-
ble that EQ metabolites were thermally degraded dur-
ing experiments [10,11].
In our in vitro studies, we observed that EQ without
and with metabolic activation with S9 mix induced
chromosome aberrations, however, in the latter case
their number was higher. Thus, a hypothesis may be
put forward that not only unchanged EQ but also
its metabolites may induce chromosome aberrations.
However, it also seems possible that the lower num-
ber of chromosome aberrations in the absence of S9
mix may be caused by cytotoxic effects of the tested
compound. The results of our studies (not presented
here) on lymphocyte viability using the trypan-blue
exclusion staining method showed that EQ at the
highest doses tested (0.25, 0.1, and 0.05 mM) with-
out metabolic activation (1-h treatment of isolated
human lymphocytes) was cytotoxic and significantly
reduced cell viability (by 42, 38, and 20%, respec-
tively). Following metabolic activation the viability
of the lymphocytes was at the control level and
was always over 95%. In the present studies, we
also observed lower cytotoxicity of EQ in experi-
ments with metabolic activation, which would have
resulted from the reaction of EQ with S9 fraction
proteins.
In our experiments, the highest numbers of aberra-
tions after EQ treatment were observed in lymphocytes
treated with the highest doses in which cytotoxicity of
the tested compound was observed (the mitotic index
was reduced, Tables 1–3). In many papers, the correla-
tions between genotoxic effect and cytotoxicity of the
test compound were discussed [27–29]. Low-toxicity
clastogens (LTC) and high-toxicity clastogens (HTC)
were described because in many cases positive results
of chromosome aberration test in vitro were observed
only at high levels of toxicity (>50%). The analysis of
many results concerning different compounds showed
that compounds inducing chromosome aberrations at
<50% toxicity were much more likely to be posi-
tive in other genotoxicity tests, and also to produce
positive effects in in vivo genotoxicity tests [27]. It
was widely accepted that high-toxicity clastogens may
be less biologically relevant than low-toxicity ones,
so the aberrations induced by HTC are not a good
predictor of in vivo or human hazard. In the present
study, EQ induced a significant number of aberrations
at the highest doses and at >50% toxicity (24-h treat-
ment without metabolic activation), but dicentric and
translocated atypical chromosomes were observed at
doses of low-toxicity as well. In the experiments with
metabolic activation of EQ, we observed a signifi-
cant increase in the number of aberrations especially
at the two highest concentrations used, but the cyto-
toxicity observed was below 50% and the dicentrics
and translocated chromosomes were also observed at
low-toxicity.
On the other hand, our results concerning EQ
genotoxicity following metabolic activation are not
consistent with in vivo results [7,8]. After metabolic
activation with a microsomal enzyme fraction isolated
from rat liver, especially at the highest concentrations
of EQ, the strongest clastogenic effect was observed.
The results obtained by HPLC showed that after
using EQ at these concentrations in cell cultures,
both the parent compound and its metabolites were
present during a 2-h treatment. In in vivo tests on the
anti-mutagenic properties of EQ, it was observed that
in chromosome aberration test and in micronucleus
test it exhibited anti-mutagenic effect, however, by
itself it did not induce chromosome aberrations, sister
chromatid exchanges, micronuclei or dominant lethal
mutations [7,8]. This may result from the fact that
EQ is quickly metabolized in animals (rat, mouse,

128
A. Błaszczyk et al. / Mutation Research 542 (2003) 117–128
Chinese hamster) and its metabolites are not muta-
genic. It suggests that EQ is probably a direct-acting
compound. In our preliminary studies using the comet
assay (results not shown here), we observed that EQ
without its metabolic activation with S9 mix induced
DNA damages.
In conclusion, the in vitro chromosome aberration
test revealed the ability of EQ to induce chromo-
some aberrations. The dose–response relationships for
abnormal cells and aberrations were at the average
level (after 24 h incubation of lymphocytes with EQ
alone, and also after metabolic activation) but dicentric
chromosomes and translocated atypical chromosomes
were observed (even after 3 h incubation of cells with
EQ alone). Further studies of mutagenic properties of
EQ with the use of other tests for mutagenicity are
indicated.
[12] A.D. Little Inc., Chemical Evaluation Committee Draft
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