Vinyl propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. Ames (OECD 471) negative. Not all necessary strains were tested. Performed based on older version of current OECD guideline

2. In vitro mammalian CHROMOSOME ABERRATION ASSAY (OECD 473). Clear Positive with and without metabolic activation. GLP conform. Performed based on an older version of current OECD guideline

3.  Literature information: Sister-chromatid exchanges induced by vinyl esters and respective carboxylic acids in cultured human lymphocytes, Sipi et al., Mutation Research 279 (1992) 75-82. Result: positive. Good quality study, detailed information, very close to OECD guideline 473.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Dec 1993 - 02 Feb 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

No version mentioned

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

No version specified

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: in vitro mammalian Chromosomal Aberration Assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell line:
The V79 cell line (1, 2) being derived from the Chinese hamster has a
• high proliferation rate (doubling time of about 12 -16 hours )
• high plating efficiency (> 90% )
• stable karyotype (modal number of 22 chromosomes) .

Storage:
Stocks of the V79 cell line (1 ml portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant . Each batch used for cytogenetic experiments was checked for
• mycoplasma contamination
• karyotype stability
• plating efficiency (incl . vital staining)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S-9 fraction

Test concentrations with justification for top dose:

Doses: 250, 500 and 1000 µg/ml (2.5, 5.0 and 9.99 mM)

The doses were determined from appropriate pretests with cultures exposed to a wide dose range of the test article, i .e . 0 .1 µg/ml - 5000 µg/ml culture medium both without and with S-9 mix .
According to the findings of the pretests, 1000 µg/ml without S-9 mix and with metabolic activation were selected as top doses . This selection was based on the observed clastogenic effect, reduced cell count and on the mitotic index .

Vehicle / solvent:

- Vehicle/solvent used: DMSO
The stability of the test substance in the carrier DMSO and in aqua dest . over a period of 4 hours was verified analytically .
With the carrier DMSO a solution was obtained and therefore, it was not necessary to verify the homogeneity analytically .

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation 750 µg ethyl-methane-sulfonate (EMS)/ml culture medium added in a volume of 1 .0 ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (S-9 mix) 1 µg cyclophosphamide/ml culture medium added in a volume of 1 .0 ml

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Preparation of test cultures
• Logarithmically growing cultures more than 50% confluent were trypsinized (2 .5% trypsin solution and Ca-Mg-free Hanks Balanced Salt Solution HBSS) . Prior to trypsin treatment the cells were rinsed once with 5 ml Ca-Mg-free HBSS .
• This process was stopped by adding MEM supplemented with 10% FCS.
• A single suspension was prepared and about 5 ml MEM supplemented with 10% FCS and containing about 30 000 - 50 000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture .
• The Quadriperm dishes were incubated at 37°C with 5% CO2 and > 90% humidity.

TREATMENT:
24 hours after seeding and incubating the cells the medium was replaced by fresh medium . The test article, dissolved in 50 Al DMSO, was added to the culture medium with or without 1 ml S-9 mix . Concurrent negative and positive controls (see item 3 .5 .4 .) were tested in parallel .
After incubation (37°C, 5% CO2, > 90% humidity) for 4 hours the serum-free medium was replaced by MEM supplemented with 10% FCS after rinsing twice with Hanks balanced salt solution (HBSS) . Subsequently, the Quadriperm dishes were incubated again until the cells were harvested .

CELL HARVEST AND PREPARATION OF METAPHASE SPREAD:
• 2 - 3 hours prior to harvesting the cells, 0.2 µg Colcemid/ml culture medium (= 1µg Colcemid dissolved in 0.1 ml PBS/culture) was added in each chamber in
order to arrest mitosis in the metaphase .
• After incubation at 37°C the culture medium was completely removed .
• For hypotonic treatment 5 ml of a 0 .4% KC1 solution which was at 37°C was added for about 20 minutes .
• Subsequently 5 ml of fixative (methanol : glacial acetic acid/3 : 1) which was at 4°C was added and kept for at least 15 minutes and then replaced . After about another 10 minutes fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation.
• The slides were taken out of the Quadriperm chambers, briefly dripped off and than rapidly passed through a Bunsen burner flame .
• The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 ml Giemsa, 185 ml Titrisol pH 7 .2) for 10 minutes .
• After being rinsed twice in aqua dest . and clarified in xylene, the preparations were mounted in Corbit-Balsam .

EVALUATION:
Chromosome analysis:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for the test substance, negative and carrier controls or 50 cells of each culture for the positive controls . Due to the very clear increase in the number of chromosomally damaged cells, the number of analyzed metaphases was limited to 150 (without S-9 mix) or 50 (with S-9 mix) and if cells had 20 -22 chromosomes, they were analyzed for chromosome aberrations.

Slides were coded before microscopic analysis . If only a few cells were found or if the metaphases were of low quality, a chromosome analysis was not carried out .


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A mitotic index based on 1500 cells/culture was determined for all test groups that yielded metaphase cells, for the carrier and untreated controls (= negative controls) and for the positive controls .:
- Cell counts: For determination of cytotoxicity additional cell cultures (using 25 cmz plastic flasks) were treated in the same way as in the main experiment . Growth inhibition was estimated by counting the number of cells in the dose groups in comparison to the concurrent carrier control at the end of the culture period using a counting chamber .

- Cell morphology: About 3 hours after test substance treatment cultures of all test groups were checked regarding cell morphology, which is an indication of attachment of the cells to the slides .
- Treatment conditions: pH values and osmolality were measured . The solubility of the test substance in the carrier used and in the aqueous
culture medium was checked to ensure proper culturing and to avoid extreme treatment conditions.

Evaluation criteria:

->Structural chromosome aberrations
- G' and G" chromatid gap and isochromatid gap unstained regions (so-called achromatic lesions) without dislocation of the segment which appears to be separated .
- B' and B" chromatid break and chromosome break visible discontinuity in chromatid or chromosome structure with lateral or longitudinal dislocation of
the fragment .
- F' and F" chromatid fragment and chromosome fragment acentric chromosome segments which occur singly or in pairs .
- D' and D" chromatid deletion and chromosome deletion loss of a segment on the level of chromatids or chromosomes .
- m. A . multiple aberrations metaphases with 5 or more aberrations excl . gaps .
- disintegration of chromosomal structure. The chromosomes being present as particles, a chromosomal structure cannot be detected any longer.
- Exchanges (translocations) These exchange aberrations (Ex) are divided into intrachanges and interchanges :
- Int' and Int" intrachanges on the level of chromatids and chromosomes
the joining of broken ends capable of reuniting two or several chromatid regions within a chromosome, e .g ., centric ring chromosomes,pericentric inversions .
- I' and I" interchanges on the level of chromatids and chromosomes the joining of broken ends capable of reuniting two or several chromosomes. They are classified as :- symmetric interchanges ,e .g ., reciprocal translocations between nonhomologous chromosomes, centric fusions, quadriradial
structures - asymmetric interchanges , e .g ., dicentric and polycentric chromosomes, triradial and quadriradial structures .

->Numerical chromosome aberrations (so-called heteroploidies)
- Aneuploidy metaphases with absent (hypoploid ) or additional (hyperploid) chromosomes. Only hyperploid metaphases are registered .
- Euploidy changes in the number of chromo-(= polyploidy) somes by whole chromosome sets .

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF AG) .
For each group the proportion of metaphases with aberrations was calculated .
A comparison of each dose group with the solvent control group was carried out using Fisher's exact test for the hypothesis of equal proportions . This test was Bonferoni-Holm corrected over the dose groups separately for each time point and was performed one-sided . If the results of this test are significant, labels (* p< 0 .05,** p< 0 .01) were printed in the tables .

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

clear growth inhibition was observed only with S-9 mix at 1000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CHROMOSOME ANALYSIS
Assay without S-9 mix ; 18 hours harvest time
- Untreated control :
8 (4 .0%) metaphases incl . gaps
3(1 .5%) metaphases excl . gaps, i .e . 1 x 2B'2Ex ; 2 x m .A . incl . Ex
1 (0 .5%) hyperploid cell
5 (2 .4%) polyploid cells
- Carrier control :
9 (4.5%) metaphases incl . gaps
7 (3 .5%) metaphases excl . gaps, i .e . 1 x B', 1 x D", 5 x Ex
4 (1 .9%) hyperploid cell s
5 (2 .4%) polyploid cells
- 1000 µg/ml :
18 (12 .0%) metaphases incl . gap s
15 (10 .0%) metaphases excl . gaps (see tables 11 - 12)
No (0 .0%) hyperploid cell s
1 (0 .7%) polyploid cell
- 750 µg EMS/ml :
With 22 (22%) aberrant cells incl . gaps and 20 (20%) aberrant mitosis excl . gaps including 1 multiple aberrant metaphase and 16 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells .

Assay with S-9 mix ; 18 hours harvest time
- Untreated control :
21 (10 .5%) metaphases incl . gaps
10 ( 5 .0%) metaphases excl . gaps, i .e . 1 x B' ; 1 x B" ; 1 x 2B" ;4xD" ; 1xF ; 2xF "
2 (1 .0%) hyperploid cells
6 (2 .9%) polyploid cells
- Carrier control :
11 (5 .5%) metaphases incl. gaps •
4 (2 .0%) metaphases excl . gaps, i .e . 1 x B' ; 1 x B" ; 2 x D "
4 (2 .0%) hyperploid cells
3 (1 .5%) polyploid cells
- 1000 µg/ml :
33 (66 .0%) metaphases incl . gap s
32 (64 .0%) metaphases excl . gaps (see tables 13 - 14)
No (0 .0%) hyperploid cell s
No (0 .0%) polyploid cells
- 1 µg cyclophosphamide/ml :
With 31 (31%) aberrant cells incl . gaps and 28 (28%) aberrant metaphases excl . gaps including 2 multiple aberrant metaphases and 19 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells .


MITOTIC INDEX
The mitotic index based on 1500 cells per culture for the different test groups without and with metabolic activation was anlyzed: a suppression of the mitotic activity was not observed .

CELL COUNTS
A clear growth inhibition was observed only with S-9 mix at 1000 µg/ml .

CELL MORPHOLOGY
At a concentration of 1000 µg/ml with and without S-9 mix some cells apeared rounded and there was slightly reduced attachment to the slides.

TREATMENT CONDITIONS
The osmolality of the untreated control was 295 and 170 with and without S-9 mix. The osmolarity of the solvent control and all treatments was from 378 to 461 mOsm
pH ranged from 7.41 to 7.79

TEST SUBSTANCE ANALYSI S
The stability of the test substance in the carrier DMSO and in aqua dest . over a period of 4 hours was verified analytically .
With the carrier DMSO a solution was obtained and therefore, it was not necessary to verify the homogeneity analytically .

Remarks on result:

other: potent chromosomedamaging (clastogenic) agent under in vitro conditions

Conclusions:

According to the results of the present in vitro cytogenetic study (OECD guideline 473), the test substance Vinylpropionate (CAS 105-38-4) leads to a very clear and statistically significant increase in the number chromosomally damaged cells both incl . and excl. gaps with a high proportion of exchanges. This increase was much more pronounced after the addition of a metabolizing system when compared to the experimental part without S-9 mix.
The clastogenic activity of the test substance was already observed by scanning the slides of the pretest for quality control and now confirmed in the 1st main experiment. Therefore, a 2nd experiment for further confirmation including an additional sampling time as required by current guidelines was not carried out.
An increase in the number of cells containing numerical chromosomal aberrations was not demonstrated. Thus, under the experimental conditions chosen here Vinylpropionate is considered to be a potent chromosome damaging (clastogenic) agent under in vitro conditions using V79 cells .

Executive summary:

The substance Vinylpropionate (CAS 105-38-4) was assessed for its potential to induce structural and/or numerical chromosomal aberrations in V79 cells in vitro both in the presence and absence of a metabolizing system.

According to pretests for the determination of the highest experimental dose, 250 /µg/ml, 500 µg/ml and 1000 µg/ml culture medium both in the experiment with and without metabolic activation were selected.

Chromosomes were prepared 18 hours after test substance treatment, which lasted for about 4 hours . Duplicate cultures were used for all experimental groups.

About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells in a metaphase-like stage of mitosis (C-metaphase). After preparation of the chromosomes and staining with Giemsa, metaphases were analyzed for chromosomal aberrations.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line.

Both of the positive control chemicals led to the expected increase in the number of cells containing structural chromosomal aberrations.

According to the results of the present study, the test substance caused a clear increase in the number of structural aberrant metaphases incl. and excl. gaps with a high proportion of exchanges. This increase was much more pronounced after adding a metabolizing system in comparison to the experiment without S-9 mix. An increase in the frequency of cells containing numerical aberrations was not demonstrated.

Thus, under the experimental conditions chosen here Vinylpropionate is considered to be a potent chromosome damaging (clastogenic) agent in V79 cells in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not provided

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Whole blood lymphocyte cultures from a healthy male donnor, aged 34

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

0.125 - 4 mM

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

CULTURE MEDIUM: 82 ml RPM1 1640 (Gibco, Glasgow, U.K.), 15 ml fetal calf serum, 1ml L-glutamine, 1 ml phytohemagglutinin and 1 ml 5-bromodeoxyuridine

METHOD OF APPLICATION:
6 ml of growth medium and 0.3 ml of whole blood were injected in 20 ml sterile ampules. Exposure was started after preincubation by injecting 10 microliter of Vinyl Propionate diluted in acetone to give the appropiate final concentration

DURATION
- Preincubation period: 24h at 37°C
- Exposure duration: 48 h

SPINDLE INHIBITOR (cytogenetic assays): Not mentioned directly on the paper, however there is a reference to the complete method used.

NUMBER OF REPLICATIONS: 2 cultures per concentration tested

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Harvesting, slide preparation and fluorescence -plus-Giemsa staining were performed according to Norppa et al., 1985; Chromosome damage induced by vinyl acetate through in vitro formation of acetaldehyde in human lymphocytes and Chinese hamster ovary cells, Cancer Res.. 45.4816-4821.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 25-30 metaphases per duplicate culture were scored for sister chromatid exchange

DETERMINATION OF CYTOTOXICITY:
- Method: AVERAGE NUMBER OF REPLICATIONS: determined by counting first-, second-, and third-division cells counted from 100 metaphases per culture. The RI (Replication Index) was evaluated according to Böhlke et al. (1983): Cytogenetic effects of acetaldehyde in lymphocytes of Germans and Japanese: SCE, clastogenic activity, and cell cycle delay, Hum Genet., 63, 285-289.

Statistics:

SCE scores from duplicate cultures were combined and analysed statistically by the two-taillored t-test.

Key result

Species / strain:

lymphocytes: primary culture

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: 1 mM was toxic, results until 0.5 mM were reported

Vehicle controls validity:

valid

Additional information on results:

Vinyl acetate induced a dose-dependent increase in the number of Sister Chromatid Exchanges/cell. (see graph attached below)
For vinyl propionate a statistically significant (P < 0.01) effect could be seen at concentrations between 0.125 and 0.5 mM. 1.0 mM was toxic.

Conclusions:

The results of this study suggest that vinyl esters form a group of genotoxins. Vinyl-propionate (CAS 105-38-4) induced an increase in the number of sister-chromatid exchanges in concentrations from 0.125 to 0.5 mM, being cytotoxic an concentrations of 1.01 mM and higher. The authors conclude that it appears that the mutagenic effect of vinyl esters is mediated through the production of acetaldehyde.

Executive summary:

The induction of Sister Chromatid exchanges by Vinyl propionate (CAS 105-38-4) was meassured in cultured human lymphocytes. The procedure in this study was similar to OECD guideline 473. For the lympocytes cultures whole blood of a healthy male donor aged 34 was used.

The cultures were exposed during 24 h to Vinyl propionate.

Vinyl propionate induced under the conditions tested a clear dose-dependant increase in sister-chromatid exchanges with respect to solvent control at concentrations from 0.125 to 0.5 mM. At a concentrations of 1.0 mM and higher it was found to be cytotoxic.

The results reported in this paper suggest that vinyl esters form a group of genotoxins. The authors conclude that it appears that the mutagenic effect of vinyl esters is mediated through the production of acetaldehyde.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

An older version form the guideline was used and E.coli strains were not tested.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

No version specified

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus (Salmonella typhimurium strains)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 ug/plate
For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of test substance in DMSO .

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

10 µg/plate for the strains TA100, TA98, TA1535 and TA 1537 with S-9 mix

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

5 µg/plate for strains TA 100 and 1535 without S-9 mix

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

10 µg/plate for the strain TA98 without S-9 mix

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate for the strain TA1537 without S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:

- Standard plate test: Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0 .6 % agar + 0 .6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants : 0 .5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 450C, and the remaining components are added in the following order :
0 .1 ml test solution
0 .1 ml bacterial suspension
0 .5 ml S-9 mix (in tests with metabolic activation)
o r
0 .5 ml phosphate buffer (in tests without metabolic activation )
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds .

- Preincubation test: 0 .1 ml test solution, 0 .1 ml bacterial suspension and 0 .5 ml S-9 mix are incubated at 37 0 C for the duration of 20 minutes . Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx . 30 seconds .

Composition of the minimal glucose agar :
980 ml aqua dest .
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate .

After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted .


- Cell density at seeding (if applicable): titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0 .1 ml of the overnight cultures is diluted to 10-6 in each case . Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order :
0 .1 ml solvent (without and with test substance)
0 .1 ml bacterial suspension (dilution : 10-6)
0 .5 ml S-9 mi x
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx . 30 seconds . After incubation at 37° for 48 hours in the dark, the bacterial colonies are counted .


NUMBER OF REPLICATIONS: 3 plates per dose or per control

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results .

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No increase in the number of his+ revertants was observed with and without metabolic activation on any tested strain.

No bacteriotoxic effect (reduced his- background growth) was observed .

Conclusions:

In this Ames test according to OECD guideline 473, the test substance Vinylpropionate (CAS 105-38-4) was not mutagenic under the experimental conditions chosen.

Executive summary:

The substance Vinyipropionate (CAS 105-38-4) was tested for mutagenicity in the Ames test according to OECD guideline 473.

Strains : TA 1535, TA 100, TA 1537, TA 98

Dose range : 20 microgram - 5000 microgram/plate

Test conditions: Standard plate test and preincubation test both with and without metabolic activation (rat liver S-9 mix) .

Solubility : Complete solubility of the test substance in DMSO .

Toxicity : No bacteriotoxic effect was observed .

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system .

Assessment :

According to the results of the present study, the test substance Vinylpropionat is not mutagenic in the Ames test under the experimental conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in-vivo study with vinyl propionate is available to draw a conclusion on the in vivo mutagenic properties of vinyl propionate (CAS 105-38-4).

Data from the analogue vinyl acetate (CAS 108-05-4) was used on a read-across approach to fulfill this endpoint information requirement.

1. Read-across from supporting substance (analogue approach). Literature information: Mäki-Paakkanen J. & Norppa H. (1987). Induction of micronuclei by vinyl acetate in mouse bone marrow cells and cultured human lymphocytes. Mutation Research, 190, pp 41-45. Result: positive only at lethal doses. Good quality study, detailed information, very close to OECD guideline 474.

2. Read across from supporting substance (analogue approach). Literature information: Lähdetie, J. (1988). Effects of vinyl acetate and acetaldehyde on sperm morphology and meiotic micronuclei in mice. Mutation Research 202, 171-178. Result: negative. Non-guideline study on the germ cell genotoxicity, intra peritoneal injection, good quality study, detailed information.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

purchased from Fluka A.G., Buchs, Switzerland

Species:

mouse

Strain:

other: C57Bl/6

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Department of Pathology, University of Helsinki.
- Age at study initiation: 12-17 week
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: standard animal housing of the Institute of Occupational Health
- Diet: commercial feed (Tamro, Helsinki), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

Route of administration:

intraperitoneal

Vehicle:

none- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 25 to 200 mg/ml.
- Dose volume: 0.01ml/g bw

Details on exposure:

Intraperitoneal inyection of 0.01 ml/g of:
250, 500, 1000 or 2000 mg/kg b.wt. of vinyl acetate in olive oil
olive oil (control animals)
20 mg/kg b.wt. of cyclophosphamide (positive controls).

Duration of treatment / exposure:

30 hours

Frequency of treatment:

single exposure

Post exposure period:

none

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Control: 9
Positive control: 10
250 and 500 mg/kg bw: 10
1000 and 200 mg/kg bw: 14

Control animals:

yes, concurrent vehicle

Positive control(s):

9-14 animals cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg b.wt.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Slides for each animal fixed and stained with May-Griinwald and Giemsa solutions
Samples were analyzed by one microscopist from coded slides.
Polychromatic and normochromatic cells were simultaneously recorded until the score for one cell type reached 1000.

Statistics:

The micronucleus data were tested statistically by a Chi square test
Ratios of polychromatic to normochromatic erythrocytes were statistically tested according to a 1-tailed t-test.

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

seen at toxic doses only

Toxicity:

yes

Remarks:

1000 and 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mortality: 6 animals died on the 1000 mg/kg bw dose group and 8 animals died on the 2000 mg/kg bw
A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow and was statistically significant at 1000 and 2000 mg/kg.
There was no effect on the number of micronuclei in normochromatic erythrocytes.
The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose, confirming citotoxicity

Treatment	Nr. of Animals	Polychromatic cells with micronuclei (%) + SD	Normochromatic cells with micronuclei  (%) + SD	Ratio of polychromatic to normochromatic cells + SD
Control, Olive oil	9	0.60 ± 0.10	0.31 ± 0.05	1.32 ± 0.12
Positive control, cyclophosphamide 20	10	2.07 a ± 0.20	0.29 ± 0.06	1.11 + 0.15
Vinyl acetate 250 mg/kg wb	10	0.55 + 0.08	0.25 ± 0.04	1.22 + 0.13
Vinyl acetate 500 mg/kg wb	10	0.72 ± 0.10	0.23 + 0.04	0.94 b + 0.12
Vinyl acetate 1000 mg/kg wb	8 (6 died)	1.33 a ± 0.29	0.20 ± 0.04	0.67 c ± 0.11
Vinyl acetate 2000 mg/kg wb	6 (8 died)	1.57 a ± 0.19	0.18 ± 0.05	0.53 d ± 0.08

a P < 0.001, compared with the frequency in control animals, X z test.

b P < 0.05, c P < 0.01, and d P< 0.001, compared with the ratio in control animals, t-test (one-tailed).

Conclusions:

Vinyl acetate induced a dose-dependant increase in polychromatic erythrocytes in the bone marrow of male mice after 30 h of intraperitoneal injection, but only at toxic doses.

Executive summary:

A dose-dependent increase in micronucleated polychromatic erythrocytes was observed in the bone marrow of male C57B1/6 mice 30 h after a single intraperitoneal injection of vinyl acetate (250, 500, 1000 or 2000 mg/kg b.wt.; (9-14 animals per group). The effect was statistically significant at 1000 mg/kg and at 2000 mg/kg of vinyl acetate. These doses were fatal to 6 and 8 out of 14 animals of the 1000 and 200 mg/kg vinyl acetate groups respectively.

The ratio of polychromatic to normochromatic cells decreased as a function of vinyl acetate dose. Cyclophosphamide (20 mg/kg), used as a positive control chemical, induced a clear increase in micronucleated polychromatic erythrocytes. None of the treatments affected the number of micronuclei in normochromatic erythrocytes.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)trans formation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological propertie
s from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

seen at toxic doses only

Toxicity:

yes

Remarks:

1000 and 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Vinyl acetate induced a dose-dependant increase in polychromatic erythrocytes in the bone marrow of male mice after 30 h of intraperitoneal injection, but only at toxic doses.
Based on this read-across, the genetic toxicity to bone marrow cells of vinyl propionate is considered ambigous.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming

from the ester functional group. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

Vinyl acetate induced a dose-dependant increase in polychromatic erythrocytes in the bone marrow of male mice after 30 h of intraperitoneal injection, but only at toxic doses.

Based on this read-across, the genetic toxicity of vinyl propionat to bone marrow cells is considered ambigous.

Reference 3

Endpoint:

genetic toxicity in vivo, other

Remarks:

Germ cell genotoxicity: micronuclei

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: genotoxicity of Vinyl acetate on the sperm cells of mice after intraperitoneal injection
- Short description of test conditions: Mice were injected intraperitoneally with a single dose of vinyl acetate. 13 days after treatment mice were killed and the newly divided round spermatids at stage I of mouse spermatogenesis were harvested and analyzed for micronuclei
- Parameters analysed / observed: Micronuclei in spermatids

GLP compliance:

no

Type of assay:

other: genotoxicty in mammalian germ cell

Specific details on test material used for the study:

Fluka AG, Buchs, Switzerland

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: Zentralinstitut fiir Versuchstierzucht GmbH in Hannover (F.R.G.)

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Single exposure

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

750 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Vinyl Acetate 1000 mg/kg wb: 9 animals
Vinyl Acetate 750 mg/kg wb: 5 animals
Vinyl Acetate 500 and 250 mg/kg: 4 animals
Vehicle (Olive oil): 4 animals
Positive control: CP: 4 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide 75mg/kg wb and adriamycin 6 mg/kg

Tissues and cell types examined:

Preparations of 1-mm segments of seminiferous tubules. 1000 Early spermatides were analyzed per animal

Details of tissue and slide preparation:

Tissues were prepared by microdisection and stained with Hoechst 33258.

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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality at dose of 1000 mg/kg (8/9) and 750 mg/kg (1/4)

Vehicle controls validity:

valid

Positive controls validity:

valid

Meiotic micronucleous frequences in spermatides of mice 13 days after intraperitoneal injection

 

Treatment	Number of surviving mice	Frequency of micronuclei in 1000 early spermatides
Vinyl Acetate 1000 mg/kg bw	1	0.0
Vinyl Acetate 750 mg/kg bw	3	0.33 +/- 0.33
Vinyl Acetate 500 mg/kg bw	4	2.25 +/- 0.85
Vinyl Acetate 250 mg/kg bw	4	2.00 +/- 1.08
Vehicle (Olive oil)	4	2.0 +/- 0.71
Physiological saline	4	1.57 +/- 0.61
Cyclophosphamide	4	4.75 +/- 0.75 **
Adriamicyn	4	4.75 +/- 3.77 **

** p < 0.01, in comparison to physiological saline controls.

Conclusions:

Vinyl acetate is not genotoxic to germ cells in mice. It did not induce micronucleous formation on germ cells of mice after intraperitoneal injection.

Executive summary:

Genotoxicity of vinyl acetate on the sperm cells of mice after intraperitoneal injection was studied.

Vinyl acetate dose concentrations were administered in the range of 250 to 1000 mg/kg bw.

After 13 days they were sacrificed, and the frequency of meiotic micronuclei was determined in early spermatids.

Vinyl Acetate was toxic at concentration 750 and 1000 mg/kg wb (1/4 and 8/9 animals died respectively).

Vinyl acetate did not increase the frequency of meiotic micronuclei in early spematids in any concentration tested.

Vinyl acetate is not genotoxic for germ cells in the mice following peritoneal injection.

Reference 4

Endpoint:

genetic toxicity in vivo, other

Remarks:

Germ cell genotoxicity: micronuclei

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)transformation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality at dose of 1000 mg/kg (8/9) and 750 mg/kg (1/4)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Vinyl acetate is not genotoxic to germ cells in mice. It did not induce micronucleous formation on germ cells of mice after intraperitoneal injection.
Based on this read-across, Vinyl propionate does not require classification as a germ cell mutagen, and no further testing for this endpoint is required.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group. The similarities in their structures and metabolic pathways serve as the

foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

Vinyl acetate is not genotoxic to germ cells in mice. It did not induce micronucleous formation on germ cells of mice after intraperitoneal injection.

Based on this read-across, Vinyl propionate does not require classification as a germ cell mutagen, and no further testing for this endpoint is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Data from the analogue vinyl acetate (CAS 108-05-4) was used on a read-across approach to fulfill this endpoint information requirement.

The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.

The detailed justification for the analogue approach is added to section 13 of this dossier.

Additional information on analogue vinyl acetate:

In setting vinyl acetate’s harmonized CLP classification (ECHA 2017b), ECHA’s Committee for Risk Assessment (RAC) summarized its genotoxic properties and concluded the “[g]enotoxicity data on vinyl acetate metabolites are in line with the hypothesis that vinyl acetate genotoxicity is mediated by acetaldehyde” with effects limited to doses that exceed the metabolic capacity of aldehyde dehydrogenases in “site of first contact” tissues (ECHA 2011a). After analyzing all available data, the RAC report concluded that “it is unlikely that the genotoxic potential of vinyl acetate is expressed in germ cells in man” (likely due to the very low systemic bioavailability of vinyl acetate and its metabolite) and that the available data show that vinyl acetate does not need to be classified as a germ cell mutagen (ECHA 2011a, b).

For detailed analysis of the available data please refer to:

European Chemicals Agency (ECHA). 2011a. Opinion proposing harmonized classification and labelling at Community level of vinyl acetate. ECHA/RAC/DOC No CLH-O-0000001742-77-01/F. Committee for Risk Assessment (RAC). Adopted June 10.

European Chemicals Agency (ECHA). 2011b. Annex 1, Background document to the Opinion proposing harmonized classification and labelling at Community level of vinyl acetate. ECHA/RAC/DOC No CLH-O-0000001742-77-01/A1. Committee for Risk Assessment (RAC). Adopted June 10.

Justification for classification or non-classification

The available in vitro information indicates that vinyl propionate (CAS 105-38-4) has potentially genotoxic properties.

No in-vivo study with vinyl propionate is available to draw a conclusion on the in vivo mutagenic properties of vinyl propionate.

Data from the analogue vinyl acetate (CAS 108-05-4) was used on a read-across approach to fulfill this endpoint information requirement.

Genotoxicity data on vinyl acetate metabolites are in line with the hypothesis that vinyl acetate genotoxicity is mediated by acetaldehyde” with effects limited to doses that exceed the metabolic capacity of aldehyde dehydrogenases in “site of first contact” (Sipi et al.,1992; ECHA, RAC opinion on Vinyl acetate, 2011b).

After analyzing all available data, the RAC report concluded that “it is unlikely that the genotoxic potential of vinyl acetate is expressed in germ cells in man” (likely due to the very low systemic bioavailability of vinyl acetate and its metabolite) and that the available data show that vinyl acetate does not need to be classified as a germ cell mutagen (ECHA 2011a, b).

Based on this read-across data, it is concluded that vinyl propionate does not require classification as a germ cell mutagen based on the available information and the RAC conclusion on vinyl acetate, and no further testing for this endpoint is required.