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EC number: 203-545-4
CAS number: 108-05-4
The key studies are considered to be bacterial mutation assays (McCann
et al., 1975, Jung et al., 1992; Watanabe et al., 1998), a mammalian
cell cytogenetic assay (Jantunen et al., 1986), a human cell
micronucleus assay (Budinsky et al., 2013), a human cell gene mutation
assay in the TK locus (Budinsky et al., 2013) with a supporting
investigation (ILS, 2010). A human cell gene mutation assay in the HPRT
locus is also available, although experimental methodology is limited
(Budinsky et al., 2013).
Approximately 300 carcinogens and
non-carcinogens of a variety of chemical types were tested for
mutagenicity in the Salmonella/microsome test. Bacteria were used as
sensitive indicators for DNA damage and mammalian liver extracts for
metabolic conversion of carcinogens to their active metabolic forms.
Vinyl acetate was shown to be negative in bacterial gene mutation assays
using Salmonella typhimurium TA98, TA100, TA1535 and TA1537, with and
without rat liver S-9 activation.
Thirty chemicals of various classes were
investigated for mutagenicity in a collaborative study (3 laboratories)
using Salmonella typhimurium TA102. Vinyl acetate was shown to be
negative in all 3 laboratories.
Vinyl acetate was tested in 2 separate
Twenty-two chemicals of various classes were
investigated for mutagenicity in a collaborative study (20 laboratories in
all) using Salmonella typhimurium TA102 and TA2638 or Escherichia coli
WP2/pKM101 and WP2 uvrA/pKM101. Vinyl acetate was tested in two separate laboratories
and was shown to be negative.
There was a clear dose-dependent increase in
chromatid breaks, gaps and total aberrations at
concentrations of 0.25 mM and above. Elevated
frequency of chromatid-type exchanges occurred at 1 mM. The mitotic
frequency started to decline at 0.5 mM and at 0.5 and 2 mM; less than
200 metaphases were found for analysis.
lymphocyte cultures: There
was a clear
dose-dependent increase in chromatid breaks, gaps and total aberrations.
A slight increase in chromatid exchanges was present at 0.5 and 1 mM and
for chromosome type breaks from 0.25 mM and higher. The number of
mitotic cells was reduced at 0.5 and 1 mM.
clastogenic effects of vinyl acetate were more pronounced in isolated
lymphocytes than in whole blood up to 5 mM.
Mean number of aberrations/100 cells from 2 cultures
Wholeblood lymphocyte cultures
Isolated lymphocyte cultures
Vinyl acetate at concentrations of greater
than or equal to 0.25 mM induced a dose-dependent increase in
chromatid-type aberrations and a slight elevation in chromosome-type
breaks in both human whole blood and isolated lymphoctyte cultures. The
clastogenic effect was more pronounced in isolated lymphocytes.
Vinyl acetate and acetaldehyde induced a
positive increase in the induction of MN at levels of vinyl acetate or acetaldehyde
exposure that induced <55±5% cytotoxicity based on relative survival of
TK6 cells compared to unexposed controls. Vinyl acetate exposure levels
of 0.25, 0.5, 1.0 and 2 mM were considered to be positive for MN
induction. Acetaldehyde exposure levels of 0.25, 0.5 and 1.0 mM were
considered to be positive for MN induction.
Under the conditions used in these
experiments, VAM and AA are mutagenic at the chromosome levels at
concentration levels =0.25 mM for 4 hours in vitro in
human TK6 cells. In vitro exposure levels to human TK6
cells of VAM and AA at concentrations =0.05 mM for four hours did not
induce chromosome damage in human cells.
The key studies are considered to be a bone marrow cytogenetic study
(Mäki-Paakanen and Norppa, 1987) and a spermatid micronucleus study
Meiotic micronucleus frequencies in early spermatids of mice 13 days
after a single injection of vinylacetate or acetaldehyde (frequency in
1000 early spermatids)
dose level (mg/kg)
number of mice
frequency (mean± SE)
0.33 ± 0.33
2.25 ± 0.85
2.00 ± 1.08
2.00 ± 0.71
1.00 ± 0.71
1.25 ± 0.48
1.5 ± 0.50
1.57 ± 0.61
4.75** ± 0.75
4.75** ± 3.77
** p<0.01 compared to saline controls
oli = olive oil, cp =cyclophosphamide, sal = saline, adm = adriamycin
Neither vinylacetate nor acetaldehyde (its
metabolite) induced micronuclei in meiotic cells.
Table 1 : Micronuclei and the ratio of
polychromatic to normochromatic erythrocytes in C57b1/6 mice bone marrow
30 h after treatment with vinyl acetate
vinyl acetate (mg/kg bw)
cyclophosphamide (positive control)
No of animals
Polychromatic cells with micronuclei (%) ± S.E.
0.60 ± 0.10
0.55** ± 0.08
0.72 ± 0.10
Normochromatic cells with micronuclei (%) ± S.E.
0.20 ± 0.04
0.29 ± 0.06
Ratio of polychromatic to normochromatic cells± S.E
1.32 ± 0.12
0.94* ± 0.12
0.67** ± 0.11
0.53*** ± 0.08
1000 polychromatic and normochromatic cells were analyzed per animal.
a = 6 died, b = 8 died.
$P < 0.001 (compared with the frequency in control animals, ¿2 test).
*P < 0.05,**P < 0.01,***P < 0.001 (compared with the ratio in control animals, 1-test (one-tailed).
dose-dependent increase in micronucleated polychromatic erythrocytes was
observed in the bone marrow
of male C57B1/6 mice 30 hr after a single intraperitoneal injection of
vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt).
The increase was statistically significant at 1000 mg/kg (1.33±0.29%
compared with 0.6±0.10%
in olive oil-treated controls) and at 2000 mg/kg (1.57±0.19%) of vinyl acetate.
The doses were fatal to 6/14 (1000 mg/kg) and 8/14 (2000 mg/kg) animals. The
ratio of polychromatic to normochromatic cells decreased with vinyl
acetate dose. The positive control substance cyclophosphamide
induced a clear increase in micronucleated
polychromatic erythrocytes (2.07 ± 0.20%). The number of micronuclei
in normochromatic erythrocytes were not affected by any treatment in the
Incorporation of the WHO/IPCS Template Mode of Action Analysis / Human
relevance framework not applicable.
IN VITRO DATA
Results have been consistently
negative in bacterial assays as reported for the three key studies that
collectively examined several Salmonella and E. Coli tester strains
(McCann et al., 1975; Jung et al., 1992; Watanabe et al., 1998). These
negative findings have been repeated in other bacterial studies (EU RAR,
2008 – including Bartsch et al., 1979; Lijinsky and Andrews, 1980;
Florin et al., 1980; Brams et al., 1987). In mammalian cells in vitro
however, vinyl acetate has been shown to induce chromosome level
mutations in two key studies, with lowest effective concentrations
(LECs) that produced significant increases in micronuclei at 250 µM
vinyl acetate in human TK6 cells (ILS, 2010), or significant increases
in chromosome aberrations in human peripheral blood lymphocytes at 200
µM vinyl acetate (Jantunen et al., 1986). Additional studies also of
human peripheral blood lymphocytes reported vinyl acetate induced
chromosome aberrations. In one multi-concentration study, the LEC found
was 200 µM (Norppa et al., 1985), while only 500 µM was tested in the
other study (Mustonen et al, 1986). In a recent in vitro micronucleus
assay (Budinsky et al, 2013) an increase in micronuclei was observed
over a 4 hour exposure period to TK6 cells, but only when rapidly
hydrolysed to acetaldehyde using conditions that favoured the
extracellular hydrolysis. In a 24 hour exposure period (where complete
VAM hydrolysis to acetaldehyde only occurred slowly) no significant
increase in micronuclei occurred suggesting rapid conversion to
acetaldehyde is required to overwhelm cellular defence mechanisms. In an
in vitro mammalian cell gene mutation assay (Kirby, 1983), increases in
mutant frequencies were reported in mouse lymphoma cells exposed to
vinyl acetate. However, a critical analysis of this study indicates that
the exposure concentrations used were several-fold greater than
stipulated in the relevant testing guideline (OECD 476) and as such,
this study is not appropriate for assessing vinyl acetate’s mutagenic
potential. Specifically, vinyl acetate concentrations as great as those
employed in the Kirby, 1983 study likely altered pH and/or osmolality,
and produced artifactually elevated mutant frequencies in mouse lymphoma
cells. However, neither pH nor osmolality were monitored in the Kirby,
1983 study. These deficiencies were noted in the EU RAR, 2008. Another
in vitro mammalian cell gene mutation assay at the TK locus of TK6 cells
(Budinsky et al., 2013) indicated a positive result under conditions
that favoured both rapid or slow extracellular hydrolysis to
acetaldehyde (although the affect seen under conditions favouring rapid
hydrolysis was greater). This suggested that this endpoint may be more
responsive than the micronucleus assay. However, acetaldehyde tested
under the same conditions produced a higher mutation frequency compared
with VAM and it is likely that acetaldehyde was responsible for the
effects observed (as hydrolysed from VAM). Results
from a similar test conducted at the HPRT locus are also available in
the same publication, indicating negative responses at all test
concentrations for both VAM and acetaldehyde.
IN VIVO DATA
Although a number of studies of
vinyl acetate’s in vivo mutagenicity have been conducted, including both
somatic and germ cell studies, there is no convincing evidence that
vinyl acetate’s in vitro mutagenicity is manifested in vivo. The two key
in vivo studies of chromosome level mutations in mice that employed a
range of vinyl acetate concentrations administered intra-peritoneally
(ip) showed no increases in micronuclei in either polychromatophillic
erythrocytes (PCE) at non-lethal vinyl acetate doses (Mäki-Paakanen and
Norppa, 1987) or in meiotic micronuclei in germ cells (Lähdetie,1988) at
any vinyl acetate dose.
Mäki-Paakanen and Norppa
administered vinyl acetate doses of 250, 500, 1000 or 2000 mg/kg with
the last two doses inducing 43 and 45% lethality, respectively. While
approximately two-fold increases in micronuclei frequencies were
observed at the lethal doses, significant increases were not observed at
the non-lethal doses. The current OECD 474 guideline requires that the
highest dose should be a MTD and accordingly the increase in micronuclei
at the doses where mortality was observed are questionable.
Lähdetie employed vinyl acetate
doses of 125, 250, 500, 750 or 1000 mg/kg ip with 25 and 90% lethality
at the two highest doses, respectively; no increases in micronuclei were
seen at any vinyl acetate dose level.
Four additional in vivo studies
that assessed micronuclei induction in blood cells in mice and rats
administered vinyl acetate by inhalation or in drinking water. These
studies were also negative, although they were non-standard assays for
this mutational endpoint in that they were 90 day sub-chronic toxicity
studies that neither specified cell type nor included positive controls
(EU RAR, 2008). A non-mutational genotoxic endpoint study of vinyl
acetate reported a weak induction of sister-chromatid-exchanges (SCE) in
vivo in rats after ip injection (Takeshita et al., 1986).
Peripheral lymphocytes from a
group of 27 workers involved in polyvinyl acetate production were
analysed for the frequencies of chromosomal aberrations. No exposure
data were reported. Small increases over controls (approximately
two-fold) were seen, but the study is flawed in not having a matched
control group and not considering possible confounding factors. The
authors did not claim a positive result and in view of the results and
deficiencies, no meaningful conclusion can be drawn (EU RAR, 2008).
SUMMARY AND DISCUSSION OF
In summary, vinyl acetate is
negative for inducing mutations in vitro in bacterial systems and at the
HPRT locus of TK6 cells, but is genotoxic in vitro at the chromosome
level (clastogenic) in mammalian cells at administered concentrations of
200 to 250 µM or greater. It is also potentially genotoxic in vitro at
the TK locus of TK6 cells, although the responses observed are likely
due to the hydrolysis product/metabolite acetaldehyde. Also at the
mutagenic concentrations, the TK locus response was largely a result of
slow growth mutant phenotypes indicative of chromosomal level mutations.
Although acute exposure to vinyl acetate might induce mutations at the
site of contact, existing in vivo studies have confirmed that systemic
mutagenic activity does not occur below lethal levels of exposure.
Metabolism of vinyl acetate to
acetaldehyde may be associated with the in vitro genotoxic profile of
vinyl acetate, since acetaldehyde is negative in bacterial mutation
assays, but positive in in vitro cytogenetic assays. This mechanism of
action of vinyl acetate has been proposed by investigators reporting
chromosomal damage, DNA/protein cross-links and DNA binding (EU RAR
2008). Studies have demonstrated that the observed DNA-DNA or
DNA-protein links following vinyl acetate exposures in vitro require
metabolic conversion of vinyl acetate to acetaldehyde (Lambert et al.,
1985; Kuykendall and Bogdanffy, 1992), and that the genotoxicity of
vinyl acetate is dependent on its metabolites (Bogdanffy et al., 2001
and references therein; Albertini, R. 2013 and references therein).
Vinyl acetate is rapidly hydrolyzed in metabolically competent cells to
acetic acid and acetaldehyde (through a vinyl alcohol intermediate), a
process that is dependent on intracellular carboxylesterase (Bogdanffy
and Valentine, 2003 and references therein). Acetaldehyde is a DNA
reactive compound that forms specific DNA adducts (Matsuda et al., 1998;
Brooks and Theruvathu 2005; Theruvathu et al, 2005; Stein et al., 2006;
Wang et al., 2006; Zhang et al., 2006) and is recognized as a transient,
mutagenic metabolite of vinyl acetate (Norppa et al., 1985; Kuykendall
and Bogdanffy, 1992).
Acetaldehyde is a normal
constituent of the metabolic pathways of animals and humans, and so any
genotoxic potential is likely to be expressed only under conditions of
significant metabolic overload. While significant data exists evaluating
the genotoxicity of acetaldehyde, along with clear evidence for
threshold effects related to overload, as for vinyl acetate, there is no
convincing evidence that acetaldehyde, the apparent mediating metabolite
of vinyl acetate toxicity, induces mutagenicity in vivo in normal
Albertini, R. (2013). Vinyl acetate monomer (VAM) genotoxicity profile:
Relevance for carcinogenicity. Critical Reviews in Toxicology, 43:8,
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and alkylating metabolites of halo-ethylenes, chlorobutadienes and
dichlorobutenes produced by rodent or human liver tissues. Arch Toxicol
Bogdanffy, M.S., Plowchalk, D.R. (2001) Mode-of-action-based dosimeters
for interspecies extrapolation of vinyl acetate inhalation risk. Inhal
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cytotoxicity, mitogenesis, and genotoxicity in carcinogen risk
assessments: The case of vinyl acetate. Toxicology letters 140-141:83-98
Brams, A., Buchet, J.P., Crutzen-Fayt, M.C., De Meester, C., Lauwerys,
R., Leonard, A. (1987) A comparative study, with 40 chemicals, of the
efficiency of the Salmonella assay and the SOS chromotest (kit
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Brooks, P., Theruvathu, J. (2005). DNA adducts from acetaldehyde:
implications for alcohol-related carcinogenesis. Alcohol. 2005
Budinsky, R. et al (2013). Nonlinear Responses for Chromosome and Gene
Level Effects Induced by Vinyl Acetate Monomer and Its Metabolite,
Acetaldehyde in TK6 Cells.Environmental and Molecular Mutagenesis
Florin, I., Rutberg, L., Curvall, M., Enzell, C.R. (1980) Screening of
tobacco smoke constituents for mutagenicity using the Ames test. Toxicol
ILS, Inc. High Content Cytotoxicity and Micronucleus Assay in Human TK6
Cells Exposed to Vinyl Acetate and Acetaldehyde (2010). ILS, Inc., 601
Keystone Park Drive, Suite 100, Durham, NC 27713.
Jantunen, K., Mäki-Paakkanen, J., Norppa, H. (1986): Induction of
chromosome aberrations by styrene and vinyl acetate in cultured human
lymphocytes: dependence on erythrocytes. Mutation Res 159: 109-116.
Jung, R., Engelhart, G., Herbolt, B., Jäckh, R., Müller, W. (1992):
Collaborative study of mutagenicity with Salmonalla typhimurium TA 102.
Mutat Res 278: 265-270
Kirby, P.E. (1983): Mouse lymphoma mutagenesis
assay with 40171 (ML-NCI 78) Microbiological
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DNA-histone crosslinking by vinyl acetate and acetaldehyde.
Carcinogenesis 13: 2095-2100
Lähdetie, J. (1988): Effects of vinyl acetate and acetaldehyde on sperm
morphology and meiotic micronuclei in mice. Mutat Res 202, 171-178.
Lambert, B., Chen, Y., He, S.M., Sten, M. (1985): DNA cross-links in
human leucocytes treated with vinyl acetate and acetaldehyde in vitro.
Mutat. Res. 146: 301-303
Lijinsky, W., Andrews, A.W. (1980) Mutagenicity of vinyl compounds in
Salmonella typhimurium. Tertatog Carcinog Mutagen 1:259-267
Mäki-Paakanen, J., Norppa, H. (1987): Induction of micronuclei by vinyl
acetate in mouse bone marrow cells and cultured human lymphocytes.
Mutation Res 190: 41-4.
McCann, J., Choi, E., Yamasaki, E., Ames, B.N. (1975): Detection of
carcinogens as mutagens in the Salmonella/microsome test: Assay of 300
chemicals. Proc Natl Acad Sci 72: 5135-5139.
Mustonen, R., Kangas, J., Vuojolahti, P., Linnainmaa, K. (1986): Effects
of phenoxyacetic acids on the induction of chromosome aberration in
vitro and in vivo. Mutagenesis 1: 241-245
Norppa, H., Tursi, F., Pfäffli, P., Mäki-Paakkanen, J., Järventaus, H.
(1985): Chromosome damage induced by vinyl acetate through in vitro
formation of acetaldehyde in human lymphocytes and Chinese hamster ovary
cells. Cancer Res 45: 4816-482.
Stein, S. et al (2006). Genotoxicity of acetaldehyde- and
crotonaldehyde-induced 1,N2-propanodeoxyguanosine DNA adducts in human
cells. Mutat Res. 2006 Sep 19;608(1):1-7. Epub 2006 Jun 21.
Takeshita, T., Iijima, S., Higurashi, M. (1986): Vinyl-acetate induced
sister chromatid exchanges in murine bone marrow cells. Proc Japan Acad
Theruvathu, J. et al (2005). Polyamines stimulate the formation of
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As vinyl acetate does not express significant genotoxic activity,
classification is not warranted under GHS/CLP.
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