Vinyl chloroacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

 The in vitro genotoxicity in bacteria of the test substance was determined in accordance with the OECD Testing Guideline 471. The Ames plate incorporation method was performed at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The experiment was repeated on a separate day using fresh cultures of the bacterial tester strains and fresh test material formulations. No significant increases in the frequency of revertant colonies were observed for any bacterial strains, with any dose the material, either with or without metabolic activation. It was concluded that the substance was not mutagenic under the conditions of the test.

 

In accordance with Annex VIII of REACH, since the result of the in vitro gene mutation study was negative, the potential of vinyl chloroacetate to induce structural chromosomal aberration in vitro was determined in accordance with the OECD Guideline for Testing of Chemicals 487. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls. Three conditions were used for the study: a 4-hour exposure in the absence or presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.  The test substance was markedly toxic and did induce statistically significant increases in the frequency of binucleate cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis. As a result of the positive response further scoring was performed on the mononucleate cells to indicate whether it was due to an aneugenic or clastogenic mechanism. Since there was no marked increase in the number of mononucleate cells with micronuclei it was considered that the response was likely to be due to clastogenic activity. The substance was mutagenic under the conditions of the test.

 

In accordance with Annex VIII of REACH, Column 2, an in vitro gene mutation study in mammalian cells does not need to be conducted because of positive result was found in an in vitro cytogenicity study in mammalian cells.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 9 August 2016 to 11 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human donor
- Sex, age and number of blood donors if applicable: non-smoking male aged 30 years (preliminary testing), non-smoking male aged 26 years (main experiment).
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

4-hour exposure experiments: 37.5, 75, 150, 200, 300, 450 and 600 μg/mL.
24-hour exposure experiment: 9.38, 18.75, 37.5, 50, 75, 100 and 150 μg/mL.
Based on the outcome the preliminary toxicity testing.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture media but soluble in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 or 24 hours

SPINDLE INHIBITOR: Cytochalasin B

STAIN: 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: A minimum of 500 cells per dose level for the CBPI, 2000 binucleated cells per dose level for the presence of micronuclei

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible.

Key result

Species / strain:

lymphocytes: peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: dose-related inhibition of CBPI

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change in pH.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see attached background documents

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached background documents
- Indication whether binucleate or mononucleate where appropriate: see attached background documents

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached background documents
- Negative (solvent/vehicle) historical control data: see attached background documents

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

See attached background material.

Conclusions:

The test substance was markedly toxic and induced statistically significant increases in the frequency of binucleate cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis. As a result of the positive response further scoring was performed on the mononucleate cells to indicate whether it was due to an aneugenic or clastogenic mechanism. Since there was no marked increase in the number of mononucleate cells with micronuclei it was considered that the response was likely to be due to clastogenic activity. The substance was mutagenic under the conditions of the test.

Executive summary:

The potential of vinyl chloroacetate to induce structural chromosomal aberration in vitro was determined in accordance with the OECD Guideline for Testing of Chemicals 487.

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls. Three conditions were used for the study: a 4-hour exposure in the absence or presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity.

 

The test substance was markedly toxic and did induce statistically significant increases in the frequency of binucleate cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis. As a result of the positive response further scoring was performed on the mononucleate cells to indicate whether it was due to an aneugenic or clastogenic mechanism. Since there was no marked increase in the number of mononucleate cells with micronuclei it was considered that the response was likely to be due to clastogenic activity. The substance was mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A hydrolysis study was performed on vinyl chloroacetate, in accordance with the OECD Guideline 111, for the purposes of REACH registration. The half-lives (1.22 hours at pH 7 and 3.92 hours at pH 4) indicate that the substance is unstable and will undergo rapid hydrolysis. The rate of hydrolysis is faster at neutral and high pH ranges but still occurs at a rapid rate under acidic conditions. Therefore hydrolysis of vinyl chloroacetate is expected to occur rapidly following oral exposure. Since the hydrolysis tests were performed at 20°C, hydrolysis may be expected to occur at a faster rate following exposure on account of the higher temperature (~37 °C) in the body.  

Two main degradation products were obtained in the study and identified as acetaldehyde (CAS 75-07-0 / EC 200-836-8) and chloroacetic acid (CAS 79-11-8 / EC 201-178-4). Considering the short half-life of vinyl chloroacetate, it can be expected that the degradation products will form rapidly in the body. Therefore it is likely that the mutagenic effect of the substance will be as a result of its degradation products in vivo.

Based on the result of the toxicokinetics behaviour of vinyl chloroacetate and the well documented in vivo mutagenic effects of acetaldehyde, it is concluded that vinyl chloroacetate will be mutagenic in vivo on account of this degradation product and should receive the same proposed classification as acetaldehyde for mutagenicity: Muta. 2, H341.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Type of information:

other: Expert assessment

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: An assessment was performed based on the toxicokinetics behaviour of the substance and the available information on its degradation products

Qualifier:

no guideline followed

Principles of method if other than guideline:

An assessment was performed based on the toxicokinetics behaviour of the substance and the available information on its degradation products

GLP compliance:

no

Type of assay:

other: An assessment was performed based on the toxicokinetics behaviour of the substance and the available information on its degradation products

Sex:

not specified

Genotoxicity:

other: An assessment was performed based on the toxicokinetics behaviour of the substance and the available information on its degradation products

Toxicity:

not specified

Remarks:

An assessment was performed based on the toxicokinetics behaviour of the substance and the available information on its degradation products

Conclusions:

Based on the result of the toxicokinetics behaviour of vinyl chloroacetate and the well documented in vivo mutagenic effects of acetaldehyde, it is concluded that vinyl chloroacetate will be mutagenic in vivo on account of this degradation product and should receive the same proposed classification as acetaldehyde for mutagenicity: Muta. 2, H341.

Executive summary:

A hydrolysis study was performed on vinyl chloroacetate, in accordance with the OECD Guideline 111, for the purposes of REACH registration. The half-lives (1.22 hours at pH 7 and 3.92 hours at pH 4) indicate that the substance is unstable and will undergo rapid hydrolysis. The rate of hydrolysis is faster at neutral and high pH ranges but still occurs at a rapid rate under acidic conditions. Therefore hydrolysis of vinyl chloroacetate is expected to occur rapidly following oral exposure. Since the hydrolysis tests were performed at 20°C, hydrolysis may be expected to occur at a faster rate following exposure on account of the higher temperature (~37 °C) in the body.  

Two main degradation products were obtained in the study and identified as acetaldehyde (CAS 75-07-0 / EC 200-836-8) and chloroacetic acid (CAS 79-11-8 / EC 201-178-4). Considering the short half-life of vinyl chloroacetate, it can be expected that the degradation products will form rapidly in the body. Therefore it is likely that the mutagenic effect of the substance will be as a result of its degradation products in vivo.

Based on the result of the toxicokinetics behaviour of vinyl chloroacetate and the well documented in vivo mutagenic effects of acetaldehyde, it is concluded that vinyl chloroacetate will be mutagenic in vivo on account of this degradation product and should receive the same proposed classification as acetaldehyde for mutagenicity: Muta. 2, H341.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Based on the result of the toxicokinetics behaviour of vinyl chloroacetate and the well documented in vivo mutagenic effects of acetaldehyde, it is concluded that vinyl chloroacetate will be mutagenic in vivo on account of this degradation product and should receive the same proposed Classification as acetaldehyde for mutagenicity: Muta. 2, H341.