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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, near guideline methodology, published in peer reviewed literature, adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 mix
Test concentrations with justification for top dose:
No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
- Homogenates of rat (or human) liver (S9 mix) were added directly to petri plates to evaluate the need for metabolic activation.

Method described in detail in Ames B N , McCann J & Yamasaki E (1975). Mutation Research.



Evaluation criteria:
Number of revertants per plate (histidine revertants on a petri plate/number of micrograms tested).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Vinyl acetate was negative in bacterial gene mutation assays using Salmonella typhimurium TA98, TA100, TA1535 and TA1537, with and without rat liver S-9 activation.
Executive summary:

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, near guideline study, published in peer reviewed journal, adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only S. typhimurium TA102 used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: phenotypically characterised tester strain
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S-9 mix
Test concentrations with justification for top dose:
5 doses were used with 3 plates per dose, up to 5000 µg/plate where possible. (Limitations were cytotoxicity or precipitation).
Vinyl acetate was tested in 3 separate laboratories.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: none reported
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: 0.025, 0.05 or 0.25 µg/plate
Positive control substance:
other: 2-aminoanthracene; 3 µg/plate
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Bacteria were grown in nutrient broth. Aliquots were frozen at -80°C. All experiments were mostly performed using the same frozen batch. For metabolic activation a 10% S9 mix from livers of Aroclor 1254-induced Sprague Dawley rats was used.



Evaluation criteria:
If a reproducible dose-dependent increase in the number of revertants was observed the chemicals were judged to be mutagenic. An increase of twice the spontaneous rate or at least 200 colonies over background were required to meet the criterion for mutagenicity.
Statistics:
No statistical analysis of the data as no appropriate statistics were suggested in the original papers.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: S. typhimurium TA 102
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Vinyl acetate showed no mutagenic potential in Salmonella typhimurium TA102 when tested in 3 different laboratories.
Executive summary:

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, guideline study, published in peer reviewed journal, adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Principles of method if other than guideline:
To assess whether the bacteria strains WP2 uvrA/pKM101 and TA102 may be included as standard tester strains in the bacterial reverse mutation assay.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium, other: TA 2638
Species / strain / cell type:
E. coli, other: WP2/pKM101
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Metabolic activation system:
Sprague-Dawley rat liver S9, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
0, 156, 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9, TA102 and TA2638
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
Remarks:
without S9, WP2/pKM101 and WP2 uvrA/pKM101
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9, all four tester strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Incubation period: 48 hrs at 37°C

NUMBER OF REPLICATIONS: 2 replicates, 3 plates per dose
Evaluation criteria:
If a reproducible dose-dependent increase in the number of revertants was observed the chemicals were judged to be mutagenic.
Statistics:
Linear regression test.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 2638
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2/pKM101
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: toxicity at 2500 µg/plate in one laboratory
Vehicle controls validity:
valid
Positive controls validity:
valid

Vinyl acetate was tested in 2 separate laboratories.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

Vinyl acetate showed no mutagenic potential in Salmonella typhimurium TA102 and TA2638 or Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 when tested in 2 different laboratories.
Executive summary:

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, near guideline study, peer reviewed literature, adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes from one 31 year old male human volunteer
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 (with L-glutamine) supplemented with foetal calf serum and phytohemagglutinin
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
24-hr treatments of 125, 250, 500, 1000 and 2000 µM vinyl acetate, acetone diluted, injection bottles
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Negative solvent / vehicle controls:
yes
Remarks:
acetone 10.8 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases from each duplicate culture (i.e. 200/treatment)

DETERMINATION OF CYTOTOXICITY
- Method: no data

NOTE: human peripheral blood lymphocytes: (met. act.: without); whole blood and isolated lymphocytes cultures; no BrdU to allow scoring only of 1st div metaphases
Evaluation criteria:
Mitotic frequencies were evaluated by scoring the number of mitotic cells and interphase nuclei (of mononuclear cells) in 1000 cells/culture (2000/treatment).
Statistics:
binomial test (one-tailed) and ¿2 test
Key result
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
LED = 250 µM in both isolated lymphocyte and whole blood cultures but ab. frequencies significantly higher in isolated lymphocyte cultures at 250 and 500 µM
Remarks on result:
other: LED = 250 µM in both isolated lymphocyte

Whole blood cultures: 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.

 

Isolated 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.

The clastogenic effects of vinyl acetate were more pronounced in isolated lymphocytes than in whole blood up to 5 mM.

 

Conc (mM)

Mitotic cells

Mean number of aberrations/100 cells from 2 cultures

Chromatid type

chromosome type

 

Total

breaks

exchanges

gaps

total gaps

breaks

rearrangements

total

gaps

included

excluded

included

excluded

Wholeblood lymphocyte cultures

control (acetone)

10.8

11.3

1.5

0

0

1.5

1.5

0

0.5

0.5

2.0

2.0

Vinylacetate

0.125

11.0

1.0

0

2.0

3.0

1.0

0.5

0

0.5

3.5

1.5

0.25

10.2

3.5

1.5

0.5

5.5*

5.0*

3.0*

0.5

3.5*

9.0**

8.5**

0.5

7.2

15.8***

2.1

8.2***

26.1***

17.9***

3.7*

0

3.7

29.7***

21.5***

1.0

4.5

41.5***

6.5***

13.5***

61.5***

48.0***

1.5

0

1.5

63.0***

49.5***

2.0

1.6

62.2**

2.6

14.5***

80.3***

65.8***

6.6*

0

6.6**

86.8***

72.3***

Isolated lymphocyte cultures

control (acetone)

10.8

9.9

0.5

0

1.0

1.5

0.5

0

1.0

1.0

2.5

1.5

Vinylacetate

0.125

11.4

1.5

0.5

2.5

2.0

4.5

0

0.5

5.0

2.5

0.25

10.3

9.0***

0.5

5.0*

9.5***

14.5***

0.5

3.5

18.0***

13.0***

0.5

8.9

26.5***

2.5*

16.5***

29.0***

45.5***

0

4.0

49.5***

33.0***

1.0

3.7

39.0***

4.5**

 

43.5***

12.5***

56.0***

0

3.5

59.5***

47.0***

*   p<0.05

** p<0.01

*** p<0.001

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation

Vinyl acetate at concentrations of =0.25mM 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.
Executive summary:

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Principles of method if other than guideline:
Reliable publication data using a method similar to the OECD 487 guideline. This allowed for micronuclei induction to be assessed in human TK6 cells when exposed to VAM and the hydrolysis product acetaldehyde under varying conditions.
GLP compliance:
not specified
Remarks:
Unknown if study conducted in accordance with GLP
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Obtained from the American Type Culture Collection: ATCC (Manassas, VA).

Cells were maintained in RPMI 1640 medium supplemented with 10% HS plus 0.1% pluronics, sodium pyruvate (0.5%) and antibiotics. The temperature was maintained at 37 ± 1°C with 6 ± 1% CO2 in air. TK 6 cells are included in the relevant testing guideline and are a standard cell line used in evaluating chemicall induced genotoxicity.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not specified although CytoB (a standard cytokinesis blocker) is not generally used when samples are evaluated for micronuclei using flow cytometric methods.
Metabolic activation:
without
Metabolic activation system:
Preliminary experiments conducted revealed near complete hydrolysis of VAM to its metabolite acetaldehyde in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% heat-inactivated horse serum (HS) without TK cells.

Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively. Other experiments were conducted and overall only the HS supplemented medium resulted in a significant hydrolytic capability.

The dependency of micronucleus formation (alongside cytotoxicity and apoptosis) were examined against the critical event of VAM hydrolysis to acetaldehyde. Although no S9 mix was used (which generally simulates CYP450 mediated metabolism in the liver), a method allowing for hydrolysis to the key expected metabolite has been included.
Test concentrations with justification for top dose:
0.001, 0.005, 0.01, 0.05, 0.25, 0.5, 1 and 2 mM were tested.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Hank's balanced salt solution for VAM.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
Analysis of VAM:
Extracellular concentrations of VAM and its metabolite acetaldehyde present at varying timepoints/conditions were analytically verified using HPLC-UV detection at 210 and 365 nm, respectively. The conditions included the RPMI medium alone or when supplemented with either HS or fetal bovine serum (FBS) both with and without TK 6 cells. Stop solutions were used to stabilise the substances which acted by inhibiting further hydrolysis and a derivitisation technique was used to prepare samples for analysis.

Experimental conditions:
The TK 6 cells in serum (HS or FB) supplemented medium, were inoculated into 12 well dishes 20 to 24 hours prior to exposure to VAM (or the metabolite acetealdehyde). The dosing solutions for VAM were prepared in Hank's balanced salt solution. A minimum of 3 replicate wells were used per concentration typically with 6-9 concentrations tested in each experiment. Once inoculated, the plates were sealed to minimise potential volatility loss of the test compound. These were then incubated with the test solutions for a further 4 or 24 hours.

Concentrations were selected based on the preliminary studies which demonstrated increased micronucleus and cytotoxicity at concentrations > 0.25 mM (0.25 and 0.5 mM tested). For VAM test concentrations were 0.001, 0.005, 0.01, 0.05, 0.25, 0.5, 1 and 2 mM. The metabolite acetaldehyde was tested at 0.005, 0.01, 0.05, 0.25, 0.5, 1 and 2 mM. These concentrations were based on the OECD guideline target of 55 ± 5% cytotoxicity being prefferable.

After the exposure periods, the contents of each well were pelleted using centrifugation, resuspended in fresh HS supplemented medium and inoculated into new 12 well plates. These were then incubated for an additional 24 hours prior to the conduct of flow cytometry which was used to assess cytotoxicity and micronucleus formation. The flow cytometer was used to analyse 20,000 (± 2,000) cells per replicate. Staining techniques were used to identify necrotic/late stage apoptotic cells. This was conducted using a fluorescent vital dye followed up by disruption of cellular membranes and staining of cellular DNA.

The survival (cytotoxicity) of treated cells was measured using absolute counting techniques (viable nuclei to bead ratios) and was expressed relevant to control values. The relative survival, % dead/dying cells (apoptosis/necrosis) and the general frequency of micronuclei were assessed simultaneously in the same cell samples.
Rationale for test conditions:
Generally standard as per OECD 487 guideline although additional analytical determinations were included and the effects for both VAM and its metabolite acetaldehyde were assessed.
Evaluation criteria:
Not fully specified although test groups were compared with control groups to determine statistical significance of any observed effects.
Statistics:
Dunnett's test was used to determine test substance concentrations where the micronucleus response was greater than the control groups.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
4 hour exposure and only in the presence of HS supplemented media. This medium assists in hydrolysing VAM to its metabolite acetaldehyde rapidly.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
4 hour exposure in the presence of FBS supplemented medium and medium without FBS/HS (i.e. low hydrolytical conversion to acetaldehyde).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
24 hour exposure in the presence of FBS supplemented medium (i.e. complete hydrolysis but at a slow rate). A positive response was observed but only at a concentration causing an unacceptable level of cytotoxicity.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Results of pilot studies:
The pilot studies conducted with both VAM and acetaldehyde showed excessive cytotoxicity following a 4 hour exposure of TK6 cells when tested > 4 mM. These consistently exceeded the limits in the guideline (i.e. 55 ± 5%). Based on this, 2 mM was chosen as the highest concentration for both the 4 and 24 hour incubations in the subsequent studies. In the 24 hour exposure groups, concentrations of both VAM and acetaldehyde increase to 5 mM as a result of hydrolysis.

Results of VAM extracellular hydrolysis to acetaldehyde:
In preliminary experiments, near complete hydrolysis of VAM to acetaldehyde was observed in RPMI medium supplemented with HS in the absence of TK6 cells. Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively.
In general there was little impact on VAM hydrolysis using RPMI medium alone and those with medium supplemented with FBS. Only the HS supplemented medium resulted in a significant hydrolytic cabability. It was determined that the differences observed between HS and FBS supplemented media allowed for an additional dose modifier. This allowed for the dependency of micronucleus formation, cytotoxicity and apoptosis to be examined against the critical event of VAM's hydrolytic conversion to acetaldehyde.

4 hour exposure study:
Two seperate studies were conducted demonstrating a concentration dependent increase in cytotoxicity, apoptosis and micronucleus induction following exposures to VAM or acetaldehyde in the HS supplemented medium. As the responses across both studies were similar, the replicates for both were averaged (6 replicates).

VAM under these test conditions (i.e. with HS supplemented medium) produced slightly fewer micronuclei (ca. 4%) than did the metabolite acetaldehyde (ca. 6%) at a concentration of 2 mM. Concentrations of 1 and 2 mM for both VAM and acetaldehyde results in ca. 50% of less relative survival. The overlap seen here of the VAM and acetaldehyde micronucleus dose response alongside the knowledge of VAM hydrolysis in the HS supplemented medium assisted in supporting the conclusion that VAM's ability to induce micronuclei formation under these exposure condictions is likely due to the extracellular formation of acetaldehyde formed during 30 minutes following VAM exposure. Based on the two studies combined, a statistically significant increase in micronuclei was not observed until 0.25 mM of VAM or acetaldehyde was added. Lower concentrations of VAM or acetaldehyde were unable to induce micronuclei formation.

The results were compared with historical control data which demonstrated that VAM or acetaldehyde concentrations < 0.05 mM were not associated with an increase of micronuclei incidences compared with historic controls. Statistical significance was inferred at a concentration of 0.25 mM and alongside a lack of such an increase up to 0.05 mM, this (i.e. 0.05 mM) was identified as the point of departure for this endpoint under the experimental conditions.

The 4 hour exposure tests were repeated using FBS supplemented medium (not facilitating hydrolysis under aqueous conditions). Under these exposure conditions, none of the VAM concentrations (including at 2 mM) caused a statistically significant increase in micronucleus frequencies over the baseline. When testing the metabolite acetaldehyde under the same conditions induced statistically significant increases in micronuclei frequencies were noted (appearing initially at 0.25 mM with a dose-related increase at higher concentrations). This was similar to that produced by acetaldehyde in HS supplemented medium. When assessing without supplemented medium, TK6 cells also showed no increases in micronuclei formation when exposed to VAM.

24 hour exposure study:
Based on preliminary analytical investigations the hydrolysis of VAM to acetaldehyde should be complete by 24 hours, even in medium without HS supplementation (although the rate would be much slower). Based on this, 24 hour exposure of VAM in FBS supplemented media was tested. Under these conditions, statistically significant micronuclei induction did not occur until a concentration of 2 mM. However, at this concentration an unacceptable level of cytotoxicity rendered the data questionable (relative survival of 69% and 6% at 2 and 5 mM VAM, respectively). The relative survival for acetaldehyde exposed groups was 10% at 2 mM. The 24 hour exposure of acetaldehyde in FBS supplemented medium showed the expected statistically significant increases in micronucleus formation starting at 0.25 mM.

Based on this, it appeared that slow conversion of VAM to acetaldehyde over the 24 hour period in FBS supplemented medium (even when completed within 24 hours) was insufficient to provide a statistically significant induction of micronuclei above controls. This suggests that the rate of exposure to acetaldehyde produced from VAM is an important factor.



Conclusions:
The publication investigated the potential for VAM and its hydrolysis product acetaldehyde to induce micronuclei formation under varying test conditions. It was concluded that formation of micronuclei was linked to the presence of the hydrolysis product acetaldehyde, and in particular to the speed of its production.

- Slow hydrolysis conditions:
In media alone, or with media supplemented by FBS, despite evidence of complete hydrolysis of VAM by 8 to 10 hours, VAM did not increase micronucleus frequencies following either a 4 hour or a 24 hour exposure period.

- Rapid hydrolysis conditions:
In media supplemented with HS it was observed that the increase in micronuclei formation resulted from conditions where a rapid conversion of VAM to its hydrolysis product within a 30 minute period could be expected; increased micronucleus frequencies observed following a 4 hour exposure.

It is possible that the conditions favouring slower hydrolysis mean that cellular defence and DNA repair mechanisms provide protection against acetaldehyde induced cellular component damage which may be overwhelmed during rapid hydrolysis.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Principles of method if other than guideline:
Reliable publication data using a method similar to the OECD 490 guideline. This allowed for gene mutations frequencies to be assessed at the TK locus of human TK6 cells when exposed to VAM and the hydrolysis product acetaldehyde under varying conditions.
GLP compliance:
not specified
Remarks:
Unknown if study conducted in accordance with GLP
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Obtained from the American Type Culture Collection: ATCC (Manassas, VA).

Cells were maintained in RPMI 1640 medium supplemented with 10% HS plus 0.1% pluronics, sodium pyruvate (0.5%) and antibiotics. The temperature was maintained at 37 ± 1°C with 6 ± 1% CO2 in air. TK 6 cells are included in the relevant testing guideline and are a standard cell line used in evaluating chemicall induced genotoxicity.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
without
Metabolic activation system:
Preliminary experiments conducted revealed near complete hydrolysis of VAM to its metabolite acetaldehyde in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% heat-inactivated horse serum (HS) without TK cells.

Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively. Other experiments were conducted and overall only the HS supplemented medium resulted in a significant hydrolytic capability.
Test concentrations with justification for top dose:
0.001, 0.005, 0.01, 0.05, 0.25, 0.5, 1, 2 and 4 mM were tested. These were selected to achieve the target (%) relative survival of 10 to 20% as specified by OECD guidance for in-vitro mammalian mutation assays.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
vehicle not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Analysis of VAM:
Extracellular concentrations of VAM and its metabolite acetaldehyde present at varying timepoints/conditions were analytically verified using HPLC-UV detection at 210 and 365 nm, respectively. The conditions included the RPMI medium alone or when supplemented with either HS or fetal bovine serum (FBS) both with and without TK 6 cells. Stop solutions were used to stabilise the substances which acted by inhibiting further hydrolysis and a derivitisation technique was used to prepare samples for analysis.

Experimental conditions:
TK6 cells were exposed to concentrations of 0.001, 0.005, 0.01, 0.05, 0.25, 0.5, 1.0, 2.0 and 4 mM of VAM or acetaldehyde for 24 hours in HS or FBS supplemented RPMI medium. The cell survival was determined by cloning of treated cells after exposure with results expressed relative to solvent controls. The cells were rinsed with HBSS and then subcultured for 4 days. This allowed for expression of the TK-/- mutant phenotype. Cells were maintained at a specififc density by daily dilution and on Day 4, TK-/- mutants were selected by growing in HS supplemented medium containing TFT.

The cloning efficiency was determined at the time of mutant selection by plating cells in medium without the TFT selective agent. Plates were further incubated for 14 days to allow for colony formation. After 14 days the plates were counted for mutation frequency determinations (normal growth mutants) and the selection plates were supplemented with TFT to maintain selection and reincubated for 7 additional days. This allowed for the growth of TK-/- slow growth mutants.

Only results of exposures resulting in >/= 10% cell survival (based on cloning efficiency) were considered biologically relevant (because of the possibility of spurious results at excessively cytotoxic concentrations).

Rationale for test conditions:
Generally standard as per OECD 490 guideline although additional analytical determinations were included and the effects for both VAM and its metabolite acetaldehyde were assessed.
Evaluation criteria:
Positive responses (those considered biologically relevant) are typically exposure concentrations that induce a dose-response increase in TK-/- mutant frequencies with an absolute frequency of >/= 1x10^-5 total mutants or a two fold increase over the vehicle control (whichever is higher depending on the vehicle control mutation frequencies).
Statistics:
One-way ANOVA followed by a Dunnett's test was used to determine the concentration causing a statistically significant increase in the mutant frequency over the vehicle controls.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
24 hour exposure in the presence of HS supplemented medium (i.e. high hydrolytical conversion to acetaldehyde).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
24 hour exposure in the presence or absence of FBS supplemented media (i.e. low hydrolytical conversion to acetaldehyde). Complete hydrolysis to acetaldehyde is still expected within the 24 hour exposure period.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Results of VAM extracellular hydrolysis to acetaldehyde:
In preliminary experiments, near complete hydrolysis of VAM to acetaldehyde was observed in RPMI medium supplemented with HS in the absence of TK6 cells. Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively. In general there was little impact on VAM hydrolysis using RPMI medium alone and those with medium supplemented with FBS. Only the HS supplemented medium resulted in a significant hydrolytic cabability. It was determined that the differences observed between HS and FBS supplemented media allowed for an additional dose modifier.

TK gene mutation results:
Exposure to VAM for 24 hours in HS supplemented medium produced a statistically significant increase in TK mutation frequencies starting at a concentration of 0.25 mM. Relative cell survival ranged from 94% at the lowest concentration to little survival at both 2 and 4 mM. Acetaldehyde exposed under the same conditions showed a significant elevation in total mutationfrequencies starting at a lower concentration of 0.05 mM. Relative survival rangged from 116% at the lowest concentration to ca. 13% at 2 mM with no survival at 4 mM. Mutation frequencies induced by VAM in HS supplemented medium were less than those induced by acetaldehyde under the same conditions.

VAM exposure in the presence of absence of FBS showed cytotoxicity ranging from 0 to 20% at concentrations up to 1 mM and to greater than 95% at higher concentrations (which was unacceptable). Small but significant increases in mutation frequency occured at VAM concentrations of 0.25 and 0.5 mM both in the presence or absence of FBS, respectively. This suggests that this end-point may be more responsive than the micronucleus response as VAM produced significant gene mutation frequency increases in the absence of HS which is used to drive hydrolytic conversion to acetaldehyde. Acetaldehyde tested under the same conditions produced a higher mutation frequency compared with VAM. At the mutagenic concentrations, the TK locus response was largely a result of slow growth mutant phenotypes indicative of chromosomal level mutations.





Conclusions:
The publication investigated the potential for VAM and its hydrolysis product acetaldehyde to increase mutation frequencies under varying test conditions at the TK locus. A positive result was obtained when exposing TK6 cells to VAM in both HS and FBS supplemented media (and without supplementation), although this was greater in HS supplemented media. At the mutagenic concentrations, the TK locus response was largely a result of slow growth mutant phenotypes indicative of chromosomal level mutations.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Principles of method if other than guideline:
Reliable publication data using a method similar to the OECD 476 guideline. This allowed for gene mutations frequencies to be assessed at the HPRT locus of human TK6 cells when exposed to VAM and the hydrolysis product acetaldehyde under varying conditions. As the result was negative, only limited information on the methods and results were included in the publication.
GLP compliance:
not specified
Remarks:
Unknown if study conducted in accordance with GLP
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Obtained from the American Type Culture Collection: ATCC (Manassas, VA).

Cells were maintained in RPMI 1640 medium supplemented with 10% HS plus 0.1% pluronics, sodium pyruvate (0.5%) and antibiotics. The temperature was maintained at 37 ± 1°C with 6 ± 1% CO2 in air. TK 6 cells are included in the relevant testing guideline and are a standard cell line used in evaluating chemicall induced genotoxicity.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
without
Metabolic activation system:
Preliminary experiments conducted revealed near complete hydrolysis of VAM to its metabolite acetaldehyde in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% heat-inactivated horse serum (HS) without TK cells.

Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively. Other experiments were conducted and overall only the HS supplemented medium resulted in a significant hydrolytic capability.
Test concentrations with justification for top dose:
0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1 and 2 mM were tested. These were selected to achieve the target (%) relative survival of 10 to 20% as specified by OECD guidance for in-vitro mammalian mutation assays.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
vehicle not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Analysis of VAM:
Extracellular concentrations of VAM and its metabolite acetaldehyde present at varying timepoints/conditions were analytically verified using HPLC-UV detection at 210 and 365 nm, respectively. The conditions included the RPMI medium alone or when supplemented with either HS or fetal bovine serum (FBS) both with and without TK 6 cells. Stop solutions were used to stabilise the substances which acted by inhibiting further hydrolysis and a derivitisation technique was used to prepare samples for analysis.

Full details on the HPRT assay were not included in the publication as the test was negative for both VAM and acetaldehyde.



Rationale for test conditions:
Generally standard as per OECD 476 guideline although additional analytical determinations were included and the effects for both VAM and its metabolite acetaldehyde were assessed.
Evaluation criteria:
Not specified
Statistics:
Not specified
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Negative under all test conditions for both VAM and acetaldehyde
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Results of VAM extracellular hydrolysis to acetaldehyde:
In preliminary experiments, near complete hydrolysis of VAM to acetaldehyde was observed in RPMI medium supplemented with HS in the absence of TK6 cells. Additional experiments with HS supplemented media conducted at 10, 1 and 0.05 mM VAM provided degradation half-lives of 24.2, 9.5 and 7.1 minutes, respectively. In general there was little impact on VAM hydrolysis using RPMI medium alone and those with medium supplemented with FBS. Only the HS supplemented medium resulted in a significant hydrolytic cabability. It was determined that the differences observed between HS and FBS supplemented media allowed for an additional dose modifier.

HPRT Results:
Ten replicates were evaluated at each concentration of VAM tested. The 24 hour survival at 1 and 2 mM VAM was 7.1 and 0.32%. For acetaldehyde this was 12.1 and 3.2% at the same concentrations. The average mutation frequency for controls was 6.6x10^-6 and the range for VAM treated groups was 1.1x10^-6 to 8.9x10^-6.







Conclusions:
The publication investigated the potential for VAM and its hydrolysis product acetaldehyde to increase mutation frequencies under varying test conditions at the HPRT locus. A negative result was obtained when exposing TK6 cells to varying concentrations of both VAM and acetaldehyde. .
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP. Although not specifically designed for regulatory compliance, this research study is in compliance with new OECD guideline 487
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 487 (In Vitro Mammalian Cell Micronucleus Test (MNvit) V. 5)
Principles of method if other than guideline:
Followed high content cytotoxicity and genotoxicity assay protocol developed by Litron Laboratories (Rochester, NY). This high content screening protocol has undergone inter-laboratory validation trials and is compliant with the scoring methods outlined in OECD 487. Both vinyl acetate and its metabolite acetaldehyde were tested.
GLP compliance:
no
Remarks:
the study was conducted to the highest standards of research practice, but was not audited
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Chromosome level study
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium with 10% heat inactivated horse serum plus 0.1% pluronics, sodium pyruvate and antibiotics
- Conditions: 37±1°C, with 5±1% CO2 in air
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
vinyl acetate (VA): 2.0, 1.0, 0.5, 0.25, 0.05, 0.01, 0.005, 0.001 mM
acetaldehyde (AA): 4.0, 2.0, 1.0, 0.5, 0.25, 0.05, 0.01, 0.005 mM
Vehicle / solvent:
DMSO for vinyl acetate; sterile water for acetaldehyde; serial dilutions in sterile water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (vinyl acetate) or Hank's Balanced Salt Solution (20 µL/2mL RPMI)
True negative controls:
no
Remarks:
(no non-treated cultures)
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 2 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: (12-well plates seeded with 0.8 (±0.4) x 10^5 cells/mL in 2 mL per well for 20-24 hrs prior to start of exposure)
- Exposure duration: 4 hours at 37ºC
- Expression time (cells in growth medium): 24 (±1.0) hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28 (±1.0) hours

STAIN (for cytogenetic assays): MicroFlow (in vitro) test Kit reagents; fluorescent vital dye followed by disruption of cellular membranes and staining of intracellular DNA

NUMBER OF REPLICATIONS: In triplicate, three trials

NUMBER OF CELLS EVALUATED: 20,000 (±2,000) cells

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity in this assay is reported as the percent relative survival at any treatment level as determined by the ratio of healthy nuclei to standardized counting beads. Cytotoxocity is expressed as the relative survival based on the nuclei:bead ratio of unexposed control to the treated samples. Excessive cytotoxicity was declared if cell survival was less than 40%.

OTHER EXAMINATIONS: - Determination of polyploidy: N/A- Determination of endoreplication: N/A- Other: N/A
Evaluation criteria:
Frequency of MN cells per dose group compared to the unexposed controls.
Statistics:
The analysis for an increased frequency of MN cells was based on the 95% confidence limits of the concurrent media controls. A MN increase that was greater than the mean of the concurrent media controls plus 2 standard deviations was considered a significant "positive" increase.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
not applicable
Genotoxicity:
positive
Remarks:
VA and AA both induced MN in vitro at frequencies above background at exposure concentrations = 0.25 mM x 4 hrs; VA concentrations tested were 0.001, 0.005, 0.01, 0.05, 0.25, 1.0 and 2.0 mM; AA concentrations tested were 4.0, 2.0, 1.0
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In vitro exposure levels to human TK6 cells of VA and AA at concentrations =0.05 mM for 4 hours did not induce chromosome damage in human cells. Cytotoxicity exceeded acceptable levels (>60% mortality) beginning at concentrations of 2.0 mM or greater.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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.

Conclusions:
Interpretation of results (migrated information):
positive

Positive for induction of MN under the conditions of this study.
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The key studies are considered to be a bone marrow cytogenetic study (Mäki-Paakanen and Norppa, 1987) and a spermatid micronucleus study (Lähdetie, 1988).

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: other: genotoxicity in germ cells of male mice
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP, non-guideline study, published in peer reviewed literature, adequate for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The testicular genotoxic effects of vinylacetate and its hydrolysis product, acetaldehyde, were studied in mice by analyzing the induction of meiotic micronuclei.
GLP compliance:
not specified
Type of assay:
other: genotoxicity in germ cells of male mice
Species:
mouse
Strain:
other: (C57B1/6J x C3H/He)F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: University of Helsinki, Finland (parents from Zentralinstitut fur Versuchstierzucht GmbH, Hannover, FRG) or Zentralinstitut fur Versuchstierzucht GmbH, Hannover, FRG directly.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil for vinylacetate; (cold physiological saline for acetaldehyde)
Duration of treatment / exposure:
Single ip dose
Frequency of treatment:
Single ip dose
Post exposure period:
13 days
Remarks:
Doses / Concentrations:
1000, 750, 500, 250 mg/kg bw
Basis:
other: nominal in olive oil - vinylacetate
Remarks:
Doses / Concentrations:
500, 375, 250, 125 mg/kg bw
Basis:
other: nominal in cold physiological saline - acetaldehyde
No. of animals per sex per dose:
1, 3, 4, 4, 4 for 1000, 750, 500, 250 and 0 (olive oil) mg/kg vinyl acetate. 4, 4, 4, 7 for 375, 250, 125 and 0 (saline) mg/kg acetaldehyde. 4 for each of the positive control groups.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide 75 mg/kg or adriamycin 6 mg/kg
Tissues and cell types examined:
Preparations of 1 mm segments of seminiferous tubules, representing seminiferous epithelial stages XII-I, were made from newly divided round spermatids at stage 1 of mouse spermatogenesis.
Details of tissue and slide preparation:
The mice were killed 13 days after ip injection (the time interval allowed for analysis of cells exposed at the preleptotene stage of meiosis). Newly divided round spermatids at stage 1 of mouse spermatogenesis were collected using a micro-dissection technique. Preparations of 1 mm segments of seminiferous tubules were made and stained with Hoechst 33258; these represented seminiferous epithelial stages XII-I.
Evaluation criteria:
1000 early spermatids per mouse were scored for the number of micronuclei.
Statistics:
The statistical evaluation of meiotic micronuclei was based on Poisson distribution.
Sex:
male
Genotoxicity:
negative
Remarks:
vinylacetate
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
acetaldehyde
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Meiotic micronucleus frequencies in early spermatids of mice 13 days after a single injection of vinylacetate or acetaldehyde (frequency in 1000 early spermatids)

 

treatment

vinylacetate

oli

acetaldehyde

sal

cp

adm

dose level (mg/kg)

1000

750

500

250

0

375

250

125

0

75

6

number of mice

1

3

4

4

4

4

4

4

7

4

4

frequency (mean± SE)

0

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

(range)

 

0-1

0-4

0-5

0-3

0-3

0-2

0-2

0-4

2-9

0-16

** p<0.01 compared to saline controls

oli = olive oil, cp =cyclophosphamide, sal = saline, adm = adriamycin

 

Conclusions:
Interpretation of results (migrated information): negative
Neither vinylacetate nor acetaldehyde (its hydrolysis product) induced micronuclei in meiotic cells.
Executive summary:

Neither vinylacetate nor acetaldehyde (its metabolite) induced micronuclei in meiotic cells.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, near guideline, published in peer reviewed literature, adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
C57BL
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: University of Helsinki
- Age at study initiation: 12-17 weeks
- Assigned to test groups randomly: No details
- Housing: Standard housing - no further details
- Diet: commercial feed (Tamro, Helsinki) ad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS: No details reported

IN-LIFE DATES: Not reported
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil (Farmos, Helsinki, Finland)
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 25 - 200 mg/mL
- Other: Dose volume 0.01 mL/kg
Single injections
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
25 to 200 mg/mL solutions of vinyl acetate were prepared in olive oil and administered by intraperitoneal injection (dose volume 0.01 mL/kg) to give doses of 250 - 2000 mg/kg.


Duration of treatment / exposure:
30 hr
Frequency of treatment:
Once
Post exposure period:
30 hr
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 or 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
9 - 14
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide (Laake/Farmos-Yhtyma Oy ,Turku, Finland)
- Justification for choice of positive control(s): Not reported
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg/kg
- Ampoule's containing 100 mg cyclophosphamide and 45 mg NaCl were dissolved in 5 ml of distilled water. 1 mL of this solution was then added to 9 mL of NaCl to give the final test solution (2 mg/mL).
Tissues and cell types examined:
Bone marrow - Slides for micronucleus assay were prepared from each animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Not reported

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Groups of 9-14 mice received an intraperitoneal dose of 250, 500, 1000 or 2000 mg/kg/ vinyl acetate in olive oil. The control group received an equivalent volume ( 0.01 mL/kg) of olive oil and the positive control animals received an intraperitoneal dose of cyclophosphamide (20 mg/kg). The animals were killed 30 hr after the treatment by cervical dislocation.

DETAILS OF SLIDE PREPARATION: The slides for micronucleus analysis were done according to a method described by Schmidt (1975). On the next day the preparations were stained with May-Grunwald and Giemsa solutions (Pentilla et al., 1980) with the exception that Giemsa (in 80% methanol) was purchased from Orion (Espoo, Finland).

METHOD OF ANALYSIS: The slides were analyzed by one microscopist from coded slides; 1000 polychromatic and 1000 normochromatic erythrocytes were scored per animal. The ratio of polychromatic to normochromatic cells was simultaneously recorded by counting the number of cells until the score for one cell type reached a thousand.
Statistics:
The micronucleus data were tested statistically by the ¿2 test, and the ratios of polychromatic to normochromatic erythrocytes in the in vivo assay according to a 1-tailed t-test.
Sex:
male
Genotoxicity:
ambiguous
Remarks:
seen at toxic doses only
Toxicity:
yes
Remarks:
at 1000 and 2000 mg/kg
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY:
- Induction of micronuclei (for Micronucleus assay): A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow and was statistically significant at 1000 and 2000 mg/kg, doses which were fatal to 6 and 8 out of 14 animals, respectively, in both groups (Table 1). There was no effect on the number of micronuclei in normochromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose.
- Statistical evaluation: The induction of micronucleated polychromatic erythrocytes was statistically significant at 1000 mg/kg (1.33±0.29% vs. 0.6±0.10% in olive oil-treated controls) and at 2000 mg/kg (1.57±0.19%) of vinyl acetate.

Table 1 : Micronuclei and the ratio of polychromatic to normochromatic erythrocytes in C57b1/6 mice bone marrow 30 h after treatment with vinyl acetate

Treatment

olive oil

(control)

vinyl acetate (mg/kg bw)

cyclophosphamide (positive control)

250

500

1000

2000

No of animals

9

10

10

8a

6b

10

Polychromatic cells with micronuclei (%) ± S.E.

0.60 ± 0.10

0.55** ± 0.08

0.72 ± 0.10

1.33$± 0.29

1.57$± 0.19

2.07$± 0.20

Normochromatic cells with micronuclei (%) ± S.E.

0.31± 0.05

0.25**± 0.04

0.23± 0.04

0.20 ± 0.04

0.18± 0.05

0.29 ± 0.06

Ratio of polychromatic to normochromatic cells± S.E

1.32 ± 0.12

1.22± 0.13

0.94* ± 0.12

0.67** ± 0.11

0.53*** ± 0.08

1.11± 0.1

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).

Executive summary:

A 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 (20 mg/kg) 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 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Incorporation of the WHO/IPCS Template Mode of Action Analysis / Human relevance framework not applicable.

Additional information

NON-HUMAN INFORMATION

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).

HUMAN INFORMATION

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 MUTAGENICITY

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 animals.

References:

- Albertini, R. (2013). Vinyl acetate monomer (VAM) genotoxicity profile: Relevance for carcinogenicity. Critical Reviews in Toxicology, 43:8, 671-706.

- Bartsch, H., Malaveille, C., Barbin, A., Planche, G. (1979): Mutagenic and alkylating metabolites of halo-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues. Arch Toxicol 41: 249-277

- Bogdanffy, M.S., Plowchalk, D.R. (2001) Mode-of-action-based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk. Inhal Toxicol 13:377-396

- Bogdanffy, M., Valentine, R. (2003) Differentiating between local 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 procedure). Toxicol Letters 38:123-133

- Brooks, P., Theruvathu, J. (2005). DNA adducts from acetaldehyde: implications for alcohol-related carcinogenesis. Alcohol. 2005 Apr;35(3):187-93.

- 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 54:755-768

- Florin, I., Rutberg, L., Curvall, M., Enzell, C.R. (1980) Screening of tobacco smoke constituents for mutagenicity using the Ames test. Toxicol 18:219-232

- 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 Associates; NO1-CP-1573

- Kuykendall, J.R., Bogdanffy, M.S. (1992): Reaction kinetics of 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 62: 239-242

- Theruvathu, J. et al (2005). Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde. Nucleic Acids Res. 2005 Jun 21;33(11):3513-20.

- Wang, M. et al (2006). Identification of an acetaldehyde adduct in human liver DNA and quantitation as N2-ethyldeoxyguanosine. Chem Res Toxicol. 2006 Feb;19(2):319-24.

- Watanabe, K., Sasaki, B., Kawakami, C. (1998), Comparisons of chemically-induced mutation among four bacterial strains, Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101: collaborative study III and evaluation of the usefulness of these strains. Mutation Research 416 (1998) 169-181.

Justification for classification or non-classification

As vinyl acetate does not express significant genotoxic activity, classification is not warranted under GHS/CLP.