Registration Dossier
Registration Dossier
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EC number: 902-591-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The reaction mass of methyl acetate and methanol is assessed on the basis of the individual constituents methyl acetate and methanol using a read-across approach from the supporting substances (structural analogue or surrogate).
Methyl acetate
In two in vitro gene mutation studies in bacteria according or similar to OECD guideline 471, methyl acetate was non-mutagenic to bacterial cells with and without metabolic activation.
According to ECHA disseminated dossiers, other acetates (ethyl acetate, n-butyl acetate, isobutyl acetate, n-propyl acetate) were also not mutagenic in the bacterial reverse mutation assay. Methyl acetate is rapidly hydrolysed into methanol and acetic acid after intake. Methanol did not show genetic toxicity in vitro and in vivo (see section below). There is no concern with respect to mutagenicity for acetic acid. Methyl acetate is thus not considered as a mutagen.
Methanol
In two in vitro gene mutation studies in bacteria similar to OECD guideline 471, methanol was non-mutagenic to bacterial cells with and without metabolic activation. In another in vitro gene mutation study in bacteria similar to OECD guideline 471, a slightly positive trend was indicated in one bacterial strain. In an in vitro gene mutation study in mammalian cells similar to OECD guideline 476, methanol was non-mutagenic to V79 cells with and without metabolic activation. In an in vitro micronucleus test, methanol was not clastogenic. In an in vitro DNA damage study, ambiguous results were observed with and without metabolic activation.
All in vitro studies met Klimisch score 2 criteria (study well documented, meets generally accepted scientific principles, acceptable for assessment). Based on the mainly negative in vitro results, methanol is considered to be non-mutagenic. Ambiguous results from the clastogenicity tests are disproved in several in vivo tests. Therefore methanol is also considered non-clastogenic.
Conclusion
Based on the in vitro genetic toxicity results of the two individual constituents, the reaction mass of methyl acetate and methanol is not classified for genetic toxicity.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100. Escherichia coli WP2uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from rat liver homogenate
- Test concentrations with justification for top dose:
- 0, 4, 20, 100, 500, 2500 and 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Rationale for test conditions:
- range finding test
- Evaluation criteria:
- statistically significant dose dependent increase
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Not mutagenic with or without metabolic activation
- Executive summary:
Methyl acetate was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence adn in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 micrograms/plate to 5000 micrograms/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.
Toxicity: The test compound proved to be not toxic to the bacterial strains at 5000 micrograms/plate. 5000 micrograms/plate was chosen as the top dose level for the mutagenicity study.
Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Methyl acetate did not result in relevant increases in the number of revertant colonies.
Summarizing, it can be stated that Methyl acetate is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Strains were tested with metabolic activation with 10% S-9 and then again with 30% S-9.
- Principles of method if other than guideline:
- Strains were tested with metabolic activation with 10% S-9 and then again with 30% S-9.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 97 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat and hamster liver derived S9
- Test concentrations with justification for top dose:
- 0, 100, 333, 1000, 3333, 10000 µg/Plate
- Vehicle / solvent:
- H20
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Non-mutagenic to bacterial cells
- Executive summary:
Methyl acetate was tested a 0, 100, 333, 1000, 3333 and 10000 µg/plate in Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation. Metabolic activation consisted of 10% and 30% Hamster liver derived S9 as well as 10% and 30% rat liver derived S-9 for all strains but TA 1537 which was only tested with 30% hamster- and 30% rat liver derived S9. Testing was negative with and without metabolic activation at all test levels and strains and variations of metabolic activation.
- Endpoint:
- in vitro DNA damage and/or repair study
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The endpoint of the test was cytotoxicity due to chromosome damage. The ratio of the minimal inhibitory concentration (MIC) of repair proficient to the MIC of repair-deficient E. coli strains was taken as an indicator for genotoxicity.
- GLP compliance:
- not specified
- Type of assay:
- other: DNA damage and repair assay in bacteria
- Target gene:
- not applicable
- Species / strain / cell type:
- E. coli WP2
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- other: wild-type, repair proficient
- Species / strain / cell type:
- E. coli, other: WP67
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- other: repair deficient: uvrA-, polA-
- Species / strain / cell type:
- E. coli, other: CM871
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- other: repair deficient: uvrA-, recA-, lexA-
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- No details: the initial concentration was governed by the solubility or by the toxicity of the TS as inferred from preliminary testing. Starting from this, eight 2-fold dilution steps followed in general.
- Vehicle / solvent:
- - Vehicle/solvent used: No data
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium and 2 h preincubation:
Trials were conducted in miniature form (liquid micromethod procedure: 350 μL) using microwell plates that contained nutrient broth, and as 2 h preincubation assay ("treat-and-plate method" on nutrient broth agar).
DURATION
- Preincubation period: 2 h
- Exposure duration: 16 h (liquid micromethod procedure)
DETERMINATION OF CYTOTOXICITY
- Method: other: determination of the Minimal Inhibitory Concentration (MIC): growth retardation - Evaluation criteria:
- For each tester strain, the Minimal Inhibitory Concentration (MIC) was determined. The endpoint was cytotoxicity due to chromosome damage. The ratio of the MIC of the repair proficient to the MIC of the repair deficient strains was taken as indicator for genotoxicity. A ratio of greater than 2 was accepted as significant only if reproducible in 5 parallel independent experiments.
- Key result
- Species / strain:
- E. coli, other: WP2, WP67, CM871
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 40 mg/well
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- E. coli, other: WP67, CM871
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- 20 mg/well
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 40 mg/well
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- E. coli, other: not specified
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- 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
- Species / strain:
- E. coli, other: not specified
- Metabolic activation:
- without
- Genotoxicity:
- negative
- 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:
- For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: DNA damage and/or repair
Type of study: other: DNA damage and repair assay in bacteria
Guideline:
Species/strain (Method):
- E. coli WP2
- E. coli, other: WP67
- E. coli, other: CM871 - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Remarks:
- limited documentation
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Only strains TA97 and TA102 tested
- Principles of method if other than guideline:
- According to Maron and Ames, 1983.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His-operon
- Species / strain / cell type:
- S. typhimurium TA 97
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 102
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with
- Metabolic activation system:
- S9 mix from Aroclor-induced SD rat livers
- Test concentrations with justification for top dose:
- <= 7.5 mg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Evaluation criteria:
- Positive response: dose response relationship, revertant ratio >=2.
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- not tested
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- not tested
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- not tested
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- Remarks:
- slightly positive trend
- 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:
- For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: gene mutation
Type of study: bacterial reverse mutation assay (e.g. Ames test)
Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Species/strain (Method):
- S. typhimurium TA 97
- S. typhimurium TA 102 - Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Test procedure in accordance with accepted standard methods, sufficiently documented, acceptable for assessment.
- Principles of method if other than guideline:
- In vitro micronucleus test with V79 cells comparing alcohols, acetone and various alkylating agents.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 50 µL/mL (approx. 40 mg/mL)
- Vehicle / solvent:
- - Vehicles/solvents used: medium (Eagle's MEM + 10 % FCS), acetone for positive controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- Remarks:
- 0, 0.02, 0.04, 0.08 µg/mL; solvent acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- 0, 0.4, 2, 10, 50, 100 µg/mL; solvent acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- 0, 25, 50, 100 µg/mL; solvent acetone
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 48 h
STAIN (for cytogenetic assays): 4% Giemsa
NUMBER OF REPLICATIONS: Not reported
NUMBER OF CELLS EVALUATED: 7000 interphase cells at each concentration used
OTHER
Cells were plated at a density of 13000 cells/cm², incubated for 15-18 h, then treated with the test substance. After treatment, the cells were incubated for 48 h, then collected, subjected to hypotonic treatment with KCl, fixed with acetic acid-methanol, and then stained with 4% Giemsa. - Evaluation criteria:
- Criteria used to score MN: (1) staining intensity equal to that of the nucleus, (2) diameter less than one-fifth that of the nucleus, (3) location in cytoplasm, (4) no contact with nucleus to distinguish from nuclear blebs.
- Statistics:
- The dose response was estimated by linearr regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- 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
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study, also largely meeting current standards, sufficient documentation, acceptable for assessment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Exposure and expression period combined
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- no data
- Test concentrations with justification for top dose:
- 15.8, 31.7, 47.4, 63.3 mg/mL
- Vehicle / solvent:
- - Vehicle/solvent used: culture medium (Eagle's MEM)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- Remarks:
- with S9: 1 and 2 mg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: MNNG, 0.29 and 0.59 µg/mL
- Remarks:
- without S9
- Details on test system and experimental conditions:
- DURATION
- Exposure duration: 6 days
- Expression time (cells in growth medium): Combined with 6 days exposure
- Selection time: After 6 days
SELECTION AGENT: 8-Azaguanin, 6-Thioguanin, Ouabain
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (colony formation) - Evaluation criteria:
- Significant increase in V79 cells resistant to 8-Azaguanine, 6-Thioguanine or Ouabain.
- Statistics:
- Calculation of mean mutation frequency±S.D. per 10e6 surviving cells.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 63.3 mg/ml (approx. 70 % inhibition of colony formation)
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His-operon, Trp-operon (E. coli)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 fraction of KC500-pretreated rats
- Test concentrations with justification for top dose:
- 5, 10, 50, 100, 500, 1000, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.01 and 5.0 µg/plate, respectively
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9
- Remarks:
- TA100
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- both 5.0 µg/plate, respectively
- Positive control substance:
- other: N-ethyl -N'-nitro-N-nitrosoguanidine (ENNG) without S9, 2-aminoanthracene (2AA) with S9
- Remarks:
- TA1535both 5.0 µg/plate, respectively
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.05 and 5.0 µg/plate, respectively
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, 2-aminoanthracene (2AA) with S9
- Remarks:
- WP2 uvrA
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.05 and 5.0 µg/plate, respectively
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) without S9, benzo(a)pyrene (B(a)P) with S9
- Remarks:
- TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 80.0 and 5.0 µg/plate, respectively
- Positive control substance:
- other: 9-aminoacridine (9AC) without S9, benzo(a)pyrene (B(a)P) with S9
- Remarks:
- TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.25 and 5.0 µg/plate, respectively
- Positive control substance:
- other: 4-nitroquinoline-1-oxide (4NQO) without S9, benzo(a)pyrene (B(a)P) with S9
- Remarks:
- TA1538
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: duplicates
DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition of revertant clones - Evaluation criteria:
- Doubling of revertant numbers in comparison to control and dose-response correlation.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- 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:
- For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: gene mutation
Type of study: bacterial reverse mutation assay (e.g. Ames test)
Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Species/strain (Method):
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- S. typhimurium TA 1538
- E. coli WP2 uvr A - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- limited documentation
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Salmonella TA102 and/or E.coli WP2 missing
- Principles of method if other than guideline:
- according to Ames et al., 1975.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His-operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1538
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor-induced SD rat livers
- Test concentrations with justification for top dose:
- <= 2.5 mg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- non-toxic within the range of testing
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- non-toxic within the range of testing
- 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:
- For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: Gene mutation
Type of study: Bacterial reverse mutation assay (e.g. Ames test)
Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Species/strain (Method):
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- S. typhimurium TA 1538
Referenceopen allclose all
TA100 |
||||
DOSE |
Without Activation |
With Activation |
||
µg/Plate |
Mean Value Exp 1 |
Mean Value Exp 2 |
Mean Value Exp 1 |
Mean Value Exp 2 |
0 |
168 |
129 |
168 |
148 |
4 |
178 |
137 |
178 |
136 |
20 |
170 |
133 |
170 |
137 |
100 |
169 |
140 |
169 |
146 |
500 |
185 |
139 |
185 |
135 |
2500 |
154 |
138 |
154 |
133 |
10000 |
157 |
148 |
157 |
139 |
+ ctrl |
425 |
374 |
546 |
374 |
TA1535 |
||||
DOSE |
Without Activation |
With Activation |
||
µg/Plate |
Mean Value Exp 1 |
Mean Value Exp 2 |
Mean Value Exp 1 |
Mean Value Exp 2 |
0 |
17 |
10 |
15 |
11 |
4 |
16 |
10 |
14 |
14 |
20 |
17 |
9 |
16 |
12 |
100 |
15 |
11 |
12 |
10 |
500 |
12 |
10 |
14 |
11 |
2500 |
14 |
9 |
15 |
13 |
10000 |
18 |
7 |
12 |
8 |
+ ctrl |
350 |
297 |
114 |
102 |
TA1538 |
||||
DOSE |
Without Activation |
With Activation |
||
µg/Plate |
Mean Value Exp 1 |
Mean Value Exp 2 |
Mean Value Exp 1 |
Mean Value Exp 2 |
0 |
16 |
10 |
19 |
13 |
4 |
16 |
10 |
16 |
15 |
20 |
16 |
11 |
19 |
14 |
100 |
19 |
11 |
19 |
16 |
500 |
14 |
11 |
21 |
12 |
2500 |
17 |
9 |
19 |
13 |
10000 |
14 |
9 |
19 |
15 |
+ ctrl |
439 |
376 |
491 |
348 |
TA98 |
||||
DOSE |
Without Activation |
With Activation |
||
µg/Plate |
Mean Value Exp 1 |
Mean Value Exp 2 |
Mean Value Exp 1 |
Mean Value Exp 2 |
0 |
24 |
22 |
30 |
28 |
4 |
22 |
24 |
32 |
28 |
20 |
23 |
21 |
19 |
30 |
100 |
25 |
24 |
32 |
29 |
500 |
23 |
24 |
32 |
31 |
2500 |
22 |
24 |
30 |
28 |
10000 |
24 |
24 |
31 |
28 |
+ ctrl |
342 |
402 |
471 |
487 |
TA1537 |
|
||||||||
DOSE |
|
Without Activation |
|
With Activation |
|
|
|||
µg/Plate |
|
Mean Value Exp 1 |
|
Mean Value Exp 2 |
|
Mean Value Exp 1 |
|
Mean Value Exp 2 |
|
0 |
|
11 |
|
9 |
|
11 |
|
12 |
|
4 |
|
9 |
|
8 |
|
10 |
|
10 |
|
20 |
|
11 |
|
8 |
|
10 |
|
9 |
|
100 |
|
11 |
|
9 |
|
10 |
|
10 |
|
500 |
|
9 |
|
8 |
|
9 |
|
9 |
|
2500 |
|
11 |
|
11 |
|
11 |
|
9 |
|
10000 |
|
14 |
|
7 |
|
10 |
|
8 |
|
+ ctrl |
|
98 |
|
126 |
|
98 |
|
106 |
|
WP2uvrA |
||||
DOSE |
Without Activation |
With Activation |
||
µg/Plate |
Mean Value Exp 1 |
Mean Value Exp 2 |
Mean Value Exp 1 |
Mean Value Exp 2 |
0 |
72 |
55 |
74 |
57 |
4 |
75 |
54 |
75 |
53 |
20 |
74 |
55 |
77 |
56 |
100 |
69 |
61 |
78 |
55 |
500 |
75 |
55 |
75 |
52 |
2500 |
75 |
57 |
79 |
52 |
10000 |
76 |
55 |
75 |
55 |
+ ctrl |
280 |
509 |
216 |
446 |
TA100 |
||||||||||
DOSE |
NA (-) |
10% HLI |
30% HLI |
10% RLI |
30%RLI |
|||||
µg/Plate |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
0 |
162 |
9.1 |
165 |
1.2 |
180 |
4.8 |
138 |
6.9 |
133 |
3.1 |
100.0 |
139 |
13.2 |
163 |
7.7 |
194 |
7.0 |
138 |
5.6 |
147 |
13.6 |
333.0 |
150 |
3.3 |
166 |
5.6 |
187 |
4.6 |
138 |
6.1 |
152 |
7.3 |
1000 |
150 |
5.9 |
174 |
2.5 |
179 |
6.1 |
155 |
12.1 |
157 |
4.5 |
3333 |
134 |
8.4 |
168 |
6.3 |
180 |
9.6 |
156 |
3.4 |
166 |
4.9 |
10000 |
127 |
6.5 |
162 |
5.5 |
166 |
3.8 |
149 |
5.3 |
123 |
7.2 |
POS |
412 |
8.4 |
835 |
37.2 |
545 |
10.8 |
602 |
17.8 |
842 |
33.8 |
TA1535 |
||||||||||
DOSE |
NA (-) |
10% HLI |
30% HLI |
10% RLI |
30%RLI |
|||||
µg/Plate |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
0 |
23 |
3.8 |
10 |
1.7 |
13 |
2.6 |
11 |
1.8 |
17 |
2.0 |
100.0 |
19 |
0.9 |
10 |
1.8 |
14 |
2.0 |
3 |
2.1 |
15 |
1.5 |
333.0 |
21 |
1.7 |
7 |
1.2 |
12 |
1.9 |
9 |
0.9 |
11 |
2.2 |
1000 |
20 |
2.1 |
10 |
1.0 |
17 |
2.3 |
11 |
1.0 |
12 |
1.0 |
3333 |
17 |
4.4 |
9 |
0.7 |
14 |
2.6 |
10 |
1.2 |
11 |
3.3 |
10000 |
14 |
2.5 |
9 |
2.3 |
22 |
1.5 |
8 |
0.9 |
15 |
1.3 |
POS |
562 |
16.7 |
255 |
22.5 |
403 |
13.1 |
168 |
13.3 |
96 |
2.6 |
TA97 |
||||||||||
DOSE |
NA (-) |
10% HLI |
30% HLI |
10% RLI |
30%RLI |
|||||
µg/Plate |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
0 |
194 |
4.3 |
152 |
12.5 |
208 |
3.0 |
211 |
5.5 |
199 |
13.7 |
100.0 |
174 |
15.3 |
154 |
7.8 |
217 |
3.9 |
211 |
1.9 |
221 |
9.3 |
333.0 |
145 |
13.8 |
140 |
13.9 |
200 |
10.5 |
201 |
13.0 |
213 |
7.5 |
1000 |
183 |
7.8 |
146 |
11.3 |
158 |
9.7 |
193 |
8.0 |
210 |
10.2 |
3333 |
156 |
18.5 |
141 |
12.7 |
152 |
12.1 |
209 |
5.5 |
181 |
12.4 |
10000 |
170 |
17.9 |
141 |
23.6 |
129 |
15.8 |
188 |
13.7 |
159 |
25.7 |
POS |
1158 |
52.6 |
634 |
44.5 |
330 |
13.1 |
480 |
9.2 |
404 |
1.7 |
TA98 |
||||||||||
DOSE |
NA (-) |
10% HLI |
30% HLI |
10% RLI |
30%RLI |
|||||
µg/Plate |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
0 |
15 |
2.6 |
34 |
3.2 |
32 |
2.2 |
35 |
3.2 |
33 |
3.0 |
100.0 |
18 |
3.1 |
25 |
2.8 |
29 |
3.4 |
23 |
2.8 |
34 |
3.6 |
333.0 |
14 |
2.0 |
24 |
1.2 |
27 |
0.7 |
30 |
3.5 |
32 |
5.1 |
1000 |
15 |
4.4 |
31 |
4.1 |
21 |
0.6 |
23 |
1.7 |
36 |
2.4 |
3333 |
14 |
1.5 |
27 |
3.5 |
23 |
2.3 |
25 |
2.6 |
39 |
2.1 |
10000 |
18 |
2.0 |
30 |
2.4 |
20 |
0.7 |
29 |
3.4 |
43 |
0.3 |
POS |
643 |
23.8 |
786 |
32.0 |
283 |
13.6 |
435 |
11.0 |
134 |
5.9 |
TA1537 |
||||||
DOSE |
NA (-) |
10% HLI |
30% HLI |
|||
µg/Plate |
Mean |
SEM |
Mean |
SEM |
Mean |
SEM |
0 |
8 |
1.3 |
12 |
2.3 |
11 |
0.9 |
100.0 |
4 |
0.9 |
8 |
1.3 |
10 |
1.0 |
333.0 |
6 |
0.6 |
9 |
0.7 |
9 |
3.3 |
1000 |
5 |
2.0 |
8 |
2.1 |
12 |
1.7 |
3333 |
5 |
0.6 |
8 |
2.3 |
9 |
1.5 |
10000 |
8 |
1.5 |
5 |
0.7 |
6 |
0.9 |
POS |
330 |
20.6 |
26 |
2.0 |
37 |
1.5 |
The microwell method resulted in a negative result in the presence of S9 and in a positive result in the absence of S9 at 20 mg/well (Flora et al., 1990, 1984). But the preincubation procedure was negative without S9, but ambiguous with S9 (Flora et al. 1984).
Note: The MIC concentrations were very high (40 and 20 mg/well = about 120 g/L and 60 g/L).
Given the high concentrations and the conflicting findings, it is concluded that observations made at the margin of significance (ratio = 2) are of low reliability and biological relevance. The weak relative increase of toxicity in repair-deficient strains may be an increase in unspecific cytotoxicity rather than solely "genotoxicity". (Note: A similar result was obtained with ethanol at somewhat lower concentrations).
In TA102, a slightly positive trend was indicated [(±) ambiguous], reproducible in 5 parallel independent experiments, but never exceeding the revertant ratio of 2: Spontaneous mutation rate 200 - 300/plate, while in the presence of high methanol doses, an increase in the mutation frequency above background of 140 revertants was found.
The methanol-related increase in the mutation frequency in TA102 did not fulfil the accepted criteria for mutagenic activity. Along with the high methanol doses required to induce such a weak effect and the negative results observed in all other Ames tests, the overall evidence clearly demonstrates that methanol is not mutagenic in bacterial reverse-mutation systems.
No MN increases induced by any alcohol or acetone, whereas the alkylating agents produced significant MN frequencies above medium controls.
|
Max. number of MN/1000 cells [mean±S.E.] |
|
|
Control (solvent dose) |
Chemical (dose) |
Methanol |
4.00±0.71 (50 µL/mL) |
3.50±1.19 (50 µL/mL) |
MNNG |
3.2±0.40 (5 µL/mL) |
8.2±0.83 (0.08 µg/mL) |
MMS |
3.9±0.59 (5 µL/mL) |
16.5±0.50 (100 µg/mL) |
EMS |
2.2±0.40 (5 µL/mL) |
7.0±0.69 (100 µg/mL) |
Solvent for methanol: medium
Solvent for MNNG, MMS, EMS: acetone
The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.
Maximum mutation frequency of V79 cells (per 10e6 survival cells, mean±SD) for resistance towards 6-TG, 8-AG and Ouabain after methanol treatment:
| Control | Test substance (mg/mL) | ||
Selectant | -S9 | +S9 | -S9 | +S9 |
6-Thioguanine | 0.70±1.79 | 0.94±2.18 | 1.55±3.08 (47.4 mg/mL) | 0.84±2.21 (31.7 mg/mL) |
8-Azaguanine | 23.42±8.70 | 24.29±7.30 | 22.34±7.39 (31.7 mg/mL) | 20.05±13.01 (31.7 mg/mL) |
Ouabain | 1.23±2.23 | 0 | 2.64±2.79 (47.7 mg/mL) | 0.11±0.36 (47.7 mg/mL) |
Maximum number of revertants:
| Control (water) [mean±SD] | Test substance (µg/plate) | ||
Strain | -S9 | +S9 | -S9 | +S9 |
TA100 | 149±17.1 | 161±16.2 | 175 (100) | 180 (50) |
TA1535 | 28±6.9 | 15±3.6 | 35 (50) | 17 (5000) |
TA98 | 29±6.2 | 39±8.6 | 42 (50) | 39 (1000) |
TA1537 | 16±6.4 | 21±8.1 | 22 (5000) | 35 (5) |
TA1538 | 21±5.5 | 28±7.0 | 18 (500) | 31 (5) |
E.coli WP2 uvrA | 32±7.3 | 33±10.3 | 36 (5000) | 43 (10) |
The test substance was not mutagenic in the tested strains up to 5000 µg/plate.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
The reaction mass of methyl acetate and methanol is assessed on the basis of these individual substances methyl acetate and methanol in a read-across approach.
Methyl acetate
An in vivo test according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) showed no clastogenic properties of methyl acetate. Methyl acetate is rapidly hydrolysed into methanol and acetic acid after intake. Methanol did not show in vivo genetic toxicity. There is no concern with respect to mutagenicity for acetic acid. Methyl acetate is thus not considered as a mutagen or clastogen.
Methanol
In five in vivo mammalian chromosome aberration studies, no increase in the frequency of aberrations were observed in bone marrow cells of mice after a single injection of methanol, in erythrocytes after intraperitoneal treatment with methanol for 4 days or in lung cell, spermatocytes and erythrocytes of mice after inhalative treatment for 5 days. Furthermore, methanol did no increase the frequency of micronuclei in maternal and fetal reticulocytes after treatment from gestation day 6 to 10 during pregnancy in mice. In an in vivo DNA damage/repair study, mice, rabbits and monkeys treated once or for 15 days with methanol, no increase of DNA damage was observed.
All in vivo studies met Klimisch score 2 criteria (study well documented, meets generally accepted scientific principles, acceptable for assessment). Based on the consistently negative in vivo results, methanol is considered to be non-clastogenic. As in vitro gene mutation studies were mainly negative, methanol is considered to be non-mutagenic and further in vivo testing is considered not necessary.
Conclusion
Based on the in vivo genetic toxicity results of the two individual constituents, the reaction mass of methyl acetate and methanol is not classified for genetic toxicity.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- missing positive control
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- - primary lung cells in addition to erythrocytes, not included in the guideline; no positive control (only historical)
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: C57BL/6J
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 10 weeks old
- Housing: Mice were housed in an animal facility in laminar-flow rooms
- Diet (e.g. ad libitum): Purina rodent chow
- Water (e.g. ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Air changes (per hr): 15 cycles/hour of biocleaned air
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- none
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Methanol was vaporised and transported by nitrogen and HEPA-filtered air to stainless steel chambers.
- Air flow rate: 105 L/min
- Air change rate: 15 air changes/hours
TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber methanol concentrations, monitored continuously by a Foxboro Miran 1A Infrared Analyser, indicated that ppm levels did not differ by more than 7% from calculated values. - Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- 6 h/day
- Post exposure period:
- no
- Dose / conc.:
- 5.3 mg/L air
- Dose / conc.:
- 1.04 mg/L air
- No. of animals per sex per dose:
- 5
- Control animals:
- yes
- Positive control(s):
- No positive control group was concomitantly examined. Historical positive controls are available.
- Tissues and cell types examined:
- peripheral blood; primary cultures of lung cells
- Details of tissue and slide preparation:
- Peripheral blood cell MN analysis:
Immediately following the last exposure, animals were anesthetised and blood smears were made from tail vein blood for erythrocyte MN determination. The cells were fixed in methanol and stained with acridine orange for fluorescence microscopy. All slides were coded prior to scoring of 2000 polychromatic erythrocytes (PCE) and 2000 normochromatic erythrocytes (NCE) per animal.
Lung cell MN analysis:
The same exposure groups of mice used for blood MN analysis were also used for lung cell MN analysis. After blood was removed by perfusion, the lungs were infused with a trypsin, EDTA and collagenase solution; and then removed, minced and incubated in the same enzyme solution. The cells were collected and culture dishes with 160,000 viable cells per animal were established. Lung MN were analysed in 1000 binucleated cells typically examined from each of 5 animals per dose. Percentages of mononucleated, binucleated, trinucleated and quadrinucleatexd cells were also determined. - Statistics:
- A 1- way analysis of variance was performed with Statgraphics statistical package. MN data were then analysed by a 1-tailed Dunnett´s test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not examined
- Positive controls validity:
- other: historical positive controls
- Additional information on results:
- No treatment related effect on micronuclei frequency or cell kinetics and no toxicity were seen at any dose level:
Peripheral blood cell MN analysis:
Mean MN rates were 3.1/1000 and 5.2/1000 in PCE, and 3.4/1000 and 3.6/1000 in NCE at either dose, respectively, vs. 5.0/1000 and 3.7/1000 of respective controls.
Lung cell MN analysis:
Mean MN rates were about 20/1000 to 24/1000 irrespective of a dose or control group. The ratio of bi- to mononucleated cells was not influenced by the treatment, neither was there evidence of a treatment-related increase in the incidence of multi-nucleated cells. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Principles of method if other than guideline:
- The study was performed during a developmental investigation under folate-deficient and -sufficient conditions.
After oral methanol application, the frequency of MN was examined in maternal peripheral blood and fetal blood. - GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Inc.
- Weight at study initiation: 12-14 g
- Housing: Weanling mice were housed in stainless steel, wire-bottomed cages.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23 °C
- Humidity (%): 50 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- deionised water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
2.5 g/kg (15.65 % Optima HPLC grade MeOH in deionised water) - Duration of treatment / exposure:
- gestation day 6 through 10 during pregnancy
- Frequency of treatment:
- twice daily
- Post exposure period:
- 48 h after the final exposure: maternal peripheral blood was collected
gestation day 18: fetal blood was taken - Dose / conc.:
- 2 500 mg/kg bw/day
- No. of animals per sex per dose:
- 1 to 17 dam(s)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- none
- Tissues and cell types examined:
- maternal peripheral and fetal blood
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The dose of methanol was based on previous work [Sakanashi et al., 1994, Teratology].
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Maternal vein blood (2 to 3 µL) was collected on precleaned microscope slides 48 hours (still during pregnancy) after the final methanol treatment.
Fetal blood was taken on gestation day 18.
DETAILS OF SLIDE PREPARATION:
Slides were air dried and fixed in absolute methanol on the same day. Fixed slides were stained with 0.1 % acridine orange for 5 min, followed by a 17 min rinse in Sorensen´s M/15 phosphate buffer, pH 6.8. Slides were then wet mounted with cover slips and examined by fluorescent microscopy using a mercury lamp.
METHOD OF ANALYSIS:
Micronuclei fluoresce green-yellow. The frequency of MN was scored in 1000 reticulocytes. - Statistics:
- For all statistical the pregnant dams and their litters were considered the units for comparison. Continuous variables were analysed using the two-way analysis of variance procedure and the Fisher PLSD for multiple comparisons of means. These analyses were carried out on STATVIEW SE+ (Abacus, Berkeley, CA). Incidences of frequency of micronuclei, bases on affected litters, were analyses using binomial statistics.
- Key result
- Sex:
- female
- Genotoxicity:
- negative
- Remarks:
- The frequency of micronuclei in maternal and fetal reticulocytes was not influenced by either the marginal folic acid diet (400 nmol/kg) or by methanol treatment.
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- The frequency of MN in maternal and fetal reticulocytes was similar among the groups. The MN rate was not associated with either maternal or fetal hepatic folate concentrations. There was no significant difference in the frequency of MN in reticulocytes of fetuses that were affected by malformations compared to fetuses without malformations.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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:
- yes
- Remarks:
- - limited documentation
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- Swiss Webster
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- no data
- Route of administration:
- intraperitoneal
- Vehicle:
- no data
- Details on exposure:
- no data
- Duration of treatment / exposure:
- 4 days
- Frequency of treatment:
- no data
- Post exposure period:
- 24 h
- Dose / conc.:
- 2 500 mg/kg bw/day
- Dose / conc.:
- 1 200 mg/kg bw/day
- Dose / conc.:
- 600 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Positive control(s):
- Caffeine (-folate)
Urethane (+folate) - Tissues and cell types examined:
- erythrocytes from blood samples
- Details of tissue and slide preparation:
- see any other information on materials and methods
- Evaluation criteria:
- no data
- Statistics:
- no data
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- see remarks on results
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- restriction: only one sampling time
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- - limited documentation, only one sampling time
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Route of administration:
- intraperitoneal
- Duration of treatment / exposure:
- one single injection
- Frequency of treatment:
- only one sampling time
- Post exposure period:
- 30 h
- Dose / conc.:
- 4 480 mg/kg bw/day
- Dose / conc.:
- 3 200 mg/kg bw/day
- Dose / conc.:
- 1 920 mg/kg bw/day
- No. of animals per sex per dose:
- 2
- Control animals:
- yes
- Tissues and cell types examined:
- bone-marrow cells
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- missing positive control
- Reason / purpose for cross-reference:
- reference to same study
- Principles of method if other than guideline:
- Spermatocytes of the pachytene substage of the meiotic prophase after 5 days of last exposure were isolated and morphological parameters concerning the meiotic chromosome structure were examined.
- GLP compliance:
- not specified
- Type of assay:
- chromosome aberration assay
- Species:
- mouse
- Strain:
- other: C57BL/6J
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 10 weeks old
- Housing: Mice were housed in an animal facility in laminar-flow rooms
- Diet (e.g. ad libitum): Purina rodent chow
- Water (e.g. ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Air changes (per hr): 15 cycles/hour of biocleaned air
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- none
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Methanol was vaporised and transported by nitrogen and HEPA-filtered air to stainless steel chambers.
- Air flow rate: 105 L/min
- Air change rate: 15 air changes/hours
TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber methanol concentrations, monitored continuously by a Foxboro Miran 1A Infrared Analyser, indicated that ppm levels did not differ by more than 7% from calculated values. - Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- 6 h/day
- Post exposure period:
- 5 days
- Dose / conc.:
- 5.3 mg/L air (nominal)
- Dose / conc.:
- 1.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes
- Positive control(s):
- No positive control group was concomitantly examined.
- Tissues and cell types examined:
- Spermatocytes of the pachytene substage of the meiotic prophase 5 days after last exposure
- Details of tissue and slide preparation:
- Spermatocytes of the pachytene substage of the meiotic prophase after 5 days of last exposure were isolated, and fixed for electronmicroscopy: Several morphological parameters concerning the meiotic chromosome structure were examined (synaptonemal complex analysis).
- Statistics:
- A 1- way analysis of variance was performed with Statgraphics statistical package. SC data were then analysed by a 1-tailed Dunnett´s test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- No increased frequencies of synaptonemal complex damage in spermatocytes were found.
- Toxicity:
- not specified
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- Methanol did not produce significant dose-dependent increases in SC aberrations.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- missing positive control
- Reason / purpose for cross-reference:
- reference to same study
- Principles of method if other than guideline:
- Mammalian chromosome aberration test performed with primary lung cells cultured after inhalation exposure.
- GLP compliance:
- not specified
- Type of assay:
- chromosome aberration assay
- Species:
- mouse
- Strain:
- other: C57BL/6J
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 10 weeks old
- Housing: Mice were housed in an animal facility in laminar-flow rooms
- Diet (e.g. ad libitum): Purina rodent chow
- Water (e.g. ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Air changes (per hr): 15 cycles/hour of biocleaned air
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- none
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Methanol was vaporised and transported by nitrogen and HEPA-filtered air to stainless steel chambers.
- Air flow rate: 105 L/min
- Air change rate: 15 air changes/hours
TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber methanol concentrations, monitored continuously by a Foxboro Miran 1A Infrared Analyser, indicated that ppm levels did not differ by more than 7% from calculated values. - Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- 6 h/day
- Post exposure period:
- no
- Dose / conc.:
- 5.3 mg/L air (nominal)
- Dose / conc.:
- 1.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes
- Positive control(s):
- No positive control group was concomitantly examined. Historical positive control are available.
- Tissues and cell types examined:
- primary cultures of lung cells
- Details of tissue and slide preparation:
- Immediately following the last exposure, animals were anesthetised and blood was removed by perfusion, the lungs were infused with a trypsin, EDTA and collagenase solution; and then removed, minced and incubated in the same enzyme solution. The cells were collected and culture dishes with 160,000 viable cells per animal were established. One hundred first-division metaphases per animal were examined for CA. Aberrations were classified separately by type (chromatid or chromosome) and analysed as rearrangement or breakage events. The replication indices were estimated from 200 metaphases per animal. The number of metaphases per 1000 consecutive cells was also counted for determination of mitotic indices.
- Statistics:
- A 1- way analysis of variance was performed with Statgraphics statistical package. CA data were then analysed by a 1-tailed Dunnett´s test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- There was no evidence of induced chromosome aberrations in lung cells.
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not examined
- Positive controls validity:
- other: only historical positive controls
- Additional information on results:
- No increase in the frequency of aberrations per cell and percent aberrant cells and no toxic effects were induced by the treatment:
Aberration rate per cell (including chromatid and chromosome breakage as well as rearrangement events) was from 0.04 to
0.07 in treated animals vs. 0.09 in the control group. The few aberrations found were mostly chromatid-type breaks or fragments.
Percentages of cells with abnormal chromosomes were not significantly different among treated and control groups within an experiment (6.9 % vs. 6.2 % (for 4000 ppm) and 3.8 % vs. 3.8 % (800 ppm). The replicative and mitotic indices were not significantly different from the controls. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- This study was performed to determine whether MeOH could indirectly DNA damage via ROS-mediated mechanism. Animals (mice, rabbits and monkeys) were treated with either once or for 15 days with 2.0 g/kg MeOH i.p.. Oxidative DNA damage was observed by measuring 8-oxo-2'-deoxyguanosine. Lipid peroxidation in bone marrow and spleen homogenates was determined by measuring the Ievels of HNE-His protein adducts.
- GLP compliance:
- not specified
- Remarks:
- Data from a publication, study was not conducted for regulatory purposes.
- Type of assay:
- other: oxidative DNA damage
- Species:
- other: mouse, rabbit, monkey
- Strain:
- other: CD-1, New Zealand white, cynomolgus
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: mice, rabbits and monkeys: Charles River Laboratories
- Age at study initiation: mice: 9 - 13 weeks; rabbits: 5 months; monkeys: 3.4 - 5.7 years
- Weight at study initiation: rabbits: 3.25 - 3.75 kg, monkeys: 2.8 - 4.8 kg
- Diet: mice: rodent chow ad libitum; rabbit: standard high-fiber rabbit chow; monkeys: certified primate chow diet (# 5048) from Purina Mills (St. Louis, MO) supplemented with fruit or vegetables 2-3 times weekly
- Water: mice, rabbit and monkeys: ad libitum
- Acclimation period: monkeys: two weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): mice and rabbits: 20°C; monkeys: 18 - 29°C
- Humidity (%): mice: 50%, rabbits: 60%
- Photoperiod (hrs dark / hrs light): mice: 10/14; rabbits and monkeys: 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: saline
- Details on exposure:
- Mice: Drugs were administered via intraperitoneal (ip) injection using a 26 gauge (G) 3/8 needle.
Rabbits: by ip injection usinga 23 G needle
monkeys: were lightly sedated with ketamine (ca. 5 - 10 mg/kg) for dose administration and then administered MeOH (2g/kg bw; 20% [w/v] in sterile saline) or a saline vehicle by ip injection using a 22G needle. - Duration of treatment / exposure:
- single or 15 consecutive days
- Frequency of treatment:
- daily
- Post exposure period:
- 6 hours or 15 days
- Dose / conc.:
- 2 000 mg/kg bw/day
- No. of animals per sex per dose:
- Three rabbits and primates were used in each treatment group. Four and five mice were used in each group for the acute and chronic studies, respectively.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- KBrO3
- Justification for choice of positive control: is a known renal carcinogen
- Route of administration: i.p.
- Doses / concentrations: 100 mg/kg bw at a fixed volume of 0.1 mL/10 g bw - Tissues and cell types examined:
- DNA from spleen and bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: The effect of a Iimit dose of 2.0 g/kg bw MeOH based on guidelines established for the comet assay developed in accordance with the in vivo genetic toxicology guidelines of the Organization for Econornic Co-operation and Development (OECD) were studied.
METHOD OF ANALYSIS: DNA was isolated using DNAzol, a novel guanidine-detergent lysing solution that hydrolyzes RNA and allows for the selective precipitation of DNA from cell lysates. 8-oxodG and dG were analysed via HPLC system. Lipid peroxidation in bone marrow and spleen homogenates was determined by measuring the Ievels of HNE-His protein adducts. - Statistics:
- Statistical analysis was performed using GraphPad Instat Version 3.05 (GraphPad Software, lnc., San Diego, CA). Experiments comparing two groups were analyzed by unpaired t tests and multiple comparisons were anaJyzed by one-way ANOVA followed by a Tukey post-test. The Ievel of significance was set at P<0.05.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No increase in oxidative DNA damage was observed in bone marrow or spleen at 6 h following exposure to MeOH (2.0g/kg bw ip) in mice, rabbits,
or primates. Similarly, no increase in oxidative DNA damage was observed in bone marrow or spleen 24 h following exposure to a single dose of MeOH (2.0g/kg bw ip), or following 15 consecutive daily doses of 2.0g/kg bw ip MeOH in CD-1 mice. No increase in HNE-His protein adducts was observed in bone marrow or spleen at 6 h following exposure to MeOH (2.0 g/kg bw ip) in primates. MeOH exposure (2.0 g/kg bw ip) increased HNE-His protein adducts 1.4-fold 6 h post-dose in bone marrow of mice, with adduct Ievels returning to basal values within 24 h. No increases in HNE-His protein adducts were observed in spleen of mice or bone marrow of rabbits following MeOH exposure (2.0 g/kg bw ip). MeOH exposure (2.0 g/kg bw ip) increased HNE-His protein adducts 1.5-fold 6 h post-dose in spleen of rabbits. - Conclusions:
- Interpretation of results: negative
Taken together these observations suggest that it is unlikely that exposure to MeOH would initiate Iymphomas, particularly in humans, via formation
of mutagenic oxidative DNA lesions. - Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) and EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Winklemann GmbH
- Age at study initiation: 6-7 weeks (males); 8-10 weeks (females)
- Weight at study initiation: 160-200 g
- Assigned to test groups randomly: Yes
- Housing: Makrolon cages type 4 (5 animals per cage) on soft wood granulate
- Diet: Ad libitum, rat/mice small diet ssniff(r) R/M-H (V 1534)
- Water: Ad libitum, tap water in plastic bottles
- Acclimation period: 5 days under study conditions
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/_ 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: October 13, 1998 To: December 15, 1998 - Route of administration:
- inhalation
- Vehicle:
- None
- Details on exposure:
- nose -only
- Duration of treatment / exposure:
- 28 days, 20 exposures
- Frequency of treatment:
- 6 hours/day 5 days a week
- Post exposure period:
- 24 h
- Dose / conc.:
- 0.227 mg/L air
- Dose / conc.:
- 1.057 mg/L air
- Dose / conc.:
- 6.04 mg/L air
- No. of animals per sex per dose:
- 5 male / 5 female
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- Endoxan (r), cyclophosphamide
C7H15Cl2N2P ~ H2O
50-18-0
ASTA Medica AG, Weismullerstr.45, D-60314 Frankfurt Germany
Batch No: 603575B
Administered once orally by gavage at a dose of 25 mg/kg body weight 24 hours before killing. - Tissues and cell types examined:
- Erythrocytes from femora bone marrow
- Details of tissue and slide preparation:
- Animals of the negative and postive control groups were killed by dissection of the vena cava cranialis in deep narcosis and exsanguinated. the animals of the positive control group were killed by carbon dioxide asphyxiation. For each animal, about 6 ml fetal bovine serum was poured into a centrifuge tube. one femora was removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. the mixture was then centifuged for 5 minutes at approx. 1200 rpm, after which almost all of the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Staining was performed as follows:
-5 min in methanol
- 5 min in May-Grunwald's solution
- Brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2
- Rinsing in distilled water
- Drying
- Coating with Entellan(r) - Evaluation criteria:
- 1000 polychromatic and 1000 normochromatic erythrocytes were counted for each animal. The number of cells with the micronuclei was recorded, no the number of individual micronuclei. in addition, the ratio of polychromatic erythrocytes to 1000 normochromatic erythrocytes were determined. Main parameter for the the statistical anaylsis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 1000 counted erythyrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
The test substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control gruop. A test substance producing no significant dose -related increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in the test system. - Statistics:
- A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
Based on a monotone-dose-relationship one-sided Wilcoxon test were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%. Tests on lower dose groups were only performed if all higher dose groups were significantly different from the control. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: systemic, probably secondary effects on a very low level.
- Additional information on results:
- Due to the rapid cleavage of the ester by hydrolases it was not possible to detect methyl acetat in the blood of rats (detection limit 5ppm)
- Conclusions:
- Interpretation of results (migrated information): negative
The results lead to the conclusion that methyl acetate did not lead to a substantial increase of micronucleated polychromatic erythyrocytes and is not mutagenic in the micronucleus test. - Executive summary:
The micronucleus test was carried out with methyl acetate to evaluate potential for cytogenetic damage. The animals were administered with the test compound in a rat subacute inhalation toxicity study (20 exposures within 28 days, 6 hours/day, report no. 99.0011) at exposure concentrations of 75, 350 and 2000 ppm (226.5, 1057.0 and 6040.0 mg/m3). According to the test procedure the animals were killed 24 hours after the last administration. Endoxan(r) (cyclophosphamide) was used as positive control substance and was administered once orally at a dose of 25 mg per kg body weight. The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to normochromatic erythrocytes in both and female animals remained unaffected by the treatment with Methyl acetate adn was not less than 20% of the control value. Endoxan(r) induced a marked statistically significant increase in the number of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Under the conditions of the present study the results indicate that Methyl acetate is not genotixic in the micronucleus test.
Referenceopen allclose all
Folate limitation resulted in an about 15-20 times lower folate blood level than in the high-folate groups.
4/10 folate-deficient animals receiving 2500 mg/kg died between days 2 and 3.
No difference in micronucleus frequency (MN in PCEs) and in RNA-positive blood erythrocytes was seen between treated groups and controls while animals treated with the positive control substances responded as expected:
+folate: MN 0.17 - 0.23 % vs. 0.23 % in saline control
-folate: MN 0.31 - 0.37 % Vs. 0.38 % in saline control.
Caffeine (+folate): MN 0.34 %
Caffeine (-folate): MN 2.42 %
Urethane (+folate): MN 2.52 %.
The results indicate that methanol is nonclastogenic to the developing erythroblast in bone marrow and does not inhibit red blood cell production in either normal or folate-deficient mice.
There was no significant effect on the frequency of micronuclei in treated as compared to control animals: 1.3/1000 (control and low dose), 2.0/1000 (2nd dose), and 3.7/1000 (top dose).
Sex |
Dose |
Sample Time |
Number of Animals |
Poly mean |
Normo mean |
Erythrocytes |
Eyrthrocytes with Micronuclei |
||||||||
Mean |
P/N SD |
Poly |
mean |
Mut.I |
Normo |
Mean |
Mut. I. |
||||||||
No |
% |
SD |
No |
% |
SD |
||||||||||
Pooled |
(-) Ctrl |
24H |
10 |
1000 |
1000 |
0.7 |
0.18 |
2.3 |
0.2 |
0.13 |
1.0 |
0.7 |
0.1 |
0.07 |
1.0 |
|
75 |
24H |
10 |
1000 |
1000 |
0.6 |
0.18 |
1.8 |
0.2 |
0.1 |
1.8 |
0.2 |
0.1 |
0.04 |
0.3 |
|
350 |
24H |
10 |
1000 |
1000 |
0.7 |
0.16 |
2.4 |
0.2 |
0.11 |
1.0 |
1.2 |
0.1 |
0.06 |
1.7 |
|
2000 |
24H |
10 |
1000 |
1000 |
0.6 |
0.13 |
1.6 |
0.2 |
0.1 |
0.7 |
0.8 |
0.1 |
0.08 |
1.1 |
|
(+) ctrl |
24H |
10 |
1000 |
1000 |
0.7 |
0.16 |
17.9 |
1.8 |
0.45* |
7.8 |
0.7 |
0.1 |
0.05 |
1.0 |
Sex |
Dose |
Sample Time |
Number of Animals |
Poly mean |
Normo mean |
Erythrocytes |
Eyrthrocytes with Micronuclei |
||||||||
Mean |
P/N SD |
Poly |
mean |
Mut.I |
Normo |
Mean |
Mut. I. |
||||||||
No |
% |
SD |
No |
% |
SD |
||||||||||
Male |
(-) Ctrl |
24H |
5 |
1000 |
1000 |
0.5 |
0.13 |
2.2 |
0.2 |
0.15 |
1.0 |
0.8 |
0.1 |
0.08 |
1.0 |
|
75 |
24H |
5 |
1000 |
1000 |
0.4 |
0.06 |
1.8 |
0.2 |
0.08 |
0.8 |
0.4 |
0.0 |
0.05 |
0.5 |
|
350 |
24H |
5 |
1000 |
1000 |
0.6 |
0.11 |
1.8 |
0.2 |
0.08 |
0.8 |
1.4 |
0.1 |
0.05 |
1.8 |
|
2000 |
24H |
5 |
1000 |
1000 |
0.6 |
0.11 |
1.6 |
0.2 |
0.05 |
0.7 |
1.2 |
0.1 |
0.08 |
1.5 |
|
(+) ctrl |
24H |
5 |
1000 |
1000 |
0.7 |
0.20 |
19.2 |
1.9 |
0.61 |
8.7 |
0.8 |
0.1 |
0.04 |
1.0 |
Female |
(-) Ctrl |
24H |
5 |
1000 |
1000 |
0.8 |
0.09 |
2.4 |
0.2 |
0.11 |
1.0 |
0.6 |
0.1 |
0.05 |
1.0 |
|
75 |
24H |
5 |
1000 |
1000 |
0.7 |
0.17 |
1.8 |
0.2 |
0.13 |
0.8 |
0.0 |
0.0 |
0.0 |
0.0 |
|
350 |
24H |
5 |
1000 |
1000 |
0.8 |
0.16 |
3.0 |
0.3 |
0.10 |
1.3 |
1.0 |
0.1 |
0.07 |
1.7 |
|
2000 |
24H |
5 |
1000 |
1000 |
0.7 |
0.13 |
1.6 |
0.2 |
0.13 |
0.7 |
0.4 |
0.0 |
0.05 |
0.7 |
|
(+) ctrl |
24H |
5 |
1000 |
1000 |
0.6 |
0.10 |
16.6 |
1.7 |
0.23 |
6.9 |
0.6 |
0.1 |
0.05 |
1.0 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Methyl acetate
Bacterial reverse mutation assay, RL 1
Methyl acetate was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence adn in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 micrograms/plate to 5000 micrograms/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.
Toxicity: The test compound proved to be not toxic to the bacterial strains at 5000 micrograms/plate. 5000 micrograms/plate was chosen as the top dose level for the mutagenicity study.
Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Methyl acetate did not result in relevant increases in the number of revertant colonies.
Summarizing, it can be stated that Methyl acetate is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.
Bacterial reverse mutation assay, RL2
Methyl acetate was tested a 0, 100, 333, 1000, 3333 and 10000 µg/plate in Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation. Metabolic activation consisted of 10% and 30% Hamster liver derived S9 as well as 10% and 30% rat liver derived S-9 for all strains but TA 1537 which was only tested with 30% hamster- and 30% rat liver derived S9. Testing was negative with and without metabolic activation at all test levels and strains and variations of metabolic activation.
In vivo mammalian erythrocytes micronucleus test, RL1
The test by Stammberger (1999) was performed according to OECD guideline 474. Methyl acetate was administered by inhalation (20 exposures within 28 days, 6 hours/day) to the test animals at doses of 75, 350 and 2000 ppm. The animals of the negative control group were treated with air only. The study included a concurrent positive control, which was administered once orally by gavage at a dose of 25 mg per kg body weight 24 hours before sacrifice. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity. According to the test procedure the animals were sacrificed 24 hours after the last administration. The incidence of micronucleated polychromatic erythrocytes in the dose groups of methyl acetate was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values. The positive control cyclophosphamide induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.
There is no study available in which methyl acetate was tested for mammalian cell gene mutagenicity according to OECD guideline 476. Methyl acetate is rapidly hydrolysed into methanol and acetic acid after intake. Read-across with methanol and acetic acid is therefore scientifically justified.
Methanol was tested for mammalian cell gene mutation in Chinese hamster lung fibroblasts (V79) (NEDO, 1987). This study was similar to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) (NEDO, 1987). Independent of the metabolism activation method, methanol did not induce a resistant colony (mutated colony) and did not show the gene mutation inducing property at a concentration of 31.7 to 63.3 mg/l. No increases in mutant frequency in gene mutation to drug resistance vs. neg. control was observed, whereas the pos. control DMN produced increases in dose-related manner. Only few studies are available on the mutagenicity of acetic acid (EU-RAR, 2003). Acetic acid was negative in a bacterial gene mutation test (Ames test; von der Hude et al., 1988).EFSA (2008a) concluded in the DAR for acetic acid that "Long term toxicity/carcinogenicity studies in animals with oral exposure are not necessary, considering that humans are exposed to orally ingested acetic acid from various food sources and there is no evidence that such exposure is causally related to toxic effects and an increased cancer incidence. Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay similar to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test. Although the data can be formally regarded as equivocal, they do not indicate any significant genotoxic activity for acetic anhydride in mammalian cells in vitro. This evaluation of the data reports the findings as inconclusive. Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells. Other acetates did not show mutagenic properties in the Ames assay, too; ethyl acetate (OECD SIDS, 2007a) as well as n-butyl acetate (OECD SIDS, 2008), isobutyl acetate (OECD SIDS, 2007b) and n-propyl acetate (OECD, 2009) proofed to be not mutagenic.
Conclusion: Methyl acetate was negative in bacterial reverse mutation tests and in an in vivo study according to OECD guideline 474. Furthermore, the hydrolysis products methanol and acetic acid did not reveal evidence for a mutagenic or clastogenic potential. There is no concern and no further testing needed with respect to genotoxicity. Based on the fact that methyl acetate was not mutagenic in the Ames assay and not genotoxic in the in vivo micronucleus test it is not expected that methyl acetate alters genetic material and leads to germ cell mutagenicity. The negative data is supported by data on methanol and acetic acid.
Literature:
EU-RAR (2003): EU Risk Assessment Report - Methyl Acetate, Final Report 2003
von der Hude W, Behm C, Gürtler R, Basler A (1988): Evaluation of the SOS chromo-test. Mutation Research 203, 181-194.
Morita T, Takeda K, Okumura K (1990): Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells. Mutation Research 240, 195-202.
OECD SIDS (2007a): SIDS Dossier for ethyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 14, revised May 2007.
OECD SIDS (2008): SIDS Dossier for n-butyl acetate. OECD HPV Chemical Programme, SIDS Dossier, approved at SIAM 13, revised 2008.
OECD SIDS (2007b): SIDS Dossier for isobutyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 17, dated November 2007.
OECD SIDS (2009): SIDS Dossier for n-propyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 27, dated May 2009.
EFSA (European Food Safety Authority), 2008a. Draft Assessment Report (DAR): Acetic Acid. Vol. 3, Annex B, part 2, B.6 (Aug, 2008).
Methanol
In vitro gene mutation study in bacteria, RL2
Methanol was tested in a bacterial reverse mutation study similar to OECD guideline 471, using Salmonella typhimurium TA 1535, 1537, 98, 100 and 1538 strains with and without metabolic activation. Concentrations ≤ 2.5 mg/plate were used in the plate incorporation method. Results showed no cytotoxicity within the range of tested substances and no genotoxicity with and without metabolic activation.
In vitro gene mutation study in bacteria, RL2
Methanol was tested in a bacterial reverse mutation study similar to OECD guideline 471, using Salmonella typhimurium TA 97 and 102 strains with metabolic activation. Concentrations ≤ 7.5 mg/plate were used in the plate incorporation method. In TA102, a slightly positive trend was indicated [(±) ambiguous], reproducible in 5 parallel independent experiments, but never exceeding the revertant ratio of 2: Spontaneous mutation rate 200 - 300/plate, while in the presence of high methanol doses, an increase in the mutation frequency above background of 140 revertants was found. The methanol-related increase in the mutation frequency in TA102 did not fulfil the accepted criteria for mutagenic activity. Along with the high methanol doses required to induce such a weak effect and the negative results observed in all other Ames tests, the overall evidence clearly demonstrates that methanol is not mutagenic in bacterial reverse-mutation systems.
In vitro gene mutation study in bacteria, RL2
Methanol was tested in a bacterial reverse mutation study according to OECD guideline 471, using Salmonella typhimurium TA 1535, 1537, 97, 100 and 1538 and E.coli WP2 uvrA strains with and without metabolic activation. Concentrations of 5, 10, 50, 100, 500, 1000 and 5000 µg/plate were used in the preincubation method. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, benzo[a]pyrene, N-ethyl-N’-nitro-N-nitrosoguanidine, 2-aminoanthracene, 9-aminoacridine and 4-nitroquinoline-1-oxide were used as positive controls. The test substance was not mutagenic in the tested strains up to 5000 µg/plate with and without metabolic activation.
In vitro gene mutation study in mammalian cells, RL2
Methanol was tested in a gene mutation study in mammalian cells similar to OECD guideline 476, using V79 cells with and without metabolic activation. Concentrations of 15.8, 31.7, 47.4, 63.3 mg/mL were tested. N-dimethylnitrosamine and MNNG were used a positive controls. A combined exposure and expression time of 6 days was used. Cells were selected after 6 days with 8-azaguanin, 6-thioguanin and ouabain. The test substance induced no increases in mutant frequency in gene mutation to drug resistance vs. negative control, whereas the positive control DMN produced increases in dose-related manner in the presence of metabolic activation (S9 mix), and MNNG in the absence of metabolic activation.
In vitro DNA damage/repair study in bacteria, RL2
Methanol was tested in a DNA damage/repair study in bacteria, using E.coli WP2, E.coli WP67 and E.coli CM871 strains with and without metabolic activation. The initial concentration was governed by the solubility or by the toxicity of methanol as inferred from preliminary testing. Starting from this, eight 2-fold dilution steps followed in general. Trials were conducted in miniature form (liquid micromethod procedure: 350 μL) using microwell plates that contained nutrient broth, and as 2 h preincubation assay ("treat-and-plate method" on nutrient broth agar). The exposure duration was 16 hours. Cytotoxicity was determined by determination of the Minimal Inhibitory Concentration (MIC): growth retardation. The microwell method resulted in a negative result in the presence of S9 and in a positive result in the absence of S9 at 20 mg/well (Flora et al., 1990, 1984). But the preincubation procedure was negative without S9, but ambiguous with S9 (Flora et al. 1984). Note: The MIC concentrations were very high (40 and 20 mg/well = about 120 g/L and 60 g/L). Given the high concentrations and the conflicting findings, it is concluded that observations made at the margin of significance (ratio = 2) are of low reliability and biological relevance. The weak relative increase of toxicity in repair-deficient strains may be an increase in unspecific cytotoxicity rather than solely "genotoxicity". (Note: A similar result was obtained with ethanol at somewhat lower concentrations).
In vitro micronucleus test, RL2
Methanol was tested in a gene mutation study in mammalian cells comparing alcohols, acetone and various alkylating agents using V79 cells without metabolic activation. A top dose of 50 µL/mL (approx. 40 mg/mL) was applied. N-methyl-N’-nitro-N-nitrosoguanidine (MMNG), methylmethanesulfonate and ethylmethanesulfonate were used a positive controls. Cells were plated at a density of 13000 cells/cm², incubated for 15-18 h, then treated with the test substance. After treatment, the cells were incubated for 48 h, then collected, subjected to hypotonic treatment with KCl, fixed with acetic acid-methanol, and then stained with 4% Giemsa. 7000 interphase cells were evaluated for each concentration. No MN increases were induced by any alcohol or acetone, whereas the alkylating agents produced significant MN frequencies above medium controls.
Methanol has been examined in numerous tests including bacterial, mammalian and fungal test systems. Most studies failed to demonstrate mutagenic activity, with four exceptions: 1) in Salmonella tester strain TA 102, a questionable positive response was observed (DeFlora et al., 1984a); 2) in a DNA damage and repair assay with E. coli WP strains (repair proficient and deficient types), the result was considered ambiguous (DeFlora et al. 1984b); 3) in a mouse-lymphoma assay, a significant increase in the mutation rate was reported at 7.9 mg/mL methanol (McGregor et al., 1985); and 4) in a test of mitotic chromosomal segregation in Aspergillus nidulans, a positive result was observed (Crebelli et al., 1989). All other studies produced consistently negative results (Shimizu et al., 1985; DeFlora, 1981; NEDO, 1987; Lasne et al., 1984).
In vivo chromosome aberration, RL2
A mammalian chromosome aberration test was performed with primary lung cells cultured after inhalation exposure in male mice. Methanol was vaporised with a air flow rate of 10^5 L/min and a air change rate of 15 air changes/hours. Animals were treated for 6 hours/day for 5 days without post-exposure period. 5 animals per sex per dose were used. Immediately following the last exposure, animals were anesthetised and blood was removed by perfusion, the lungs were infused with a trypsin, EDTA and collagenase solution; and then removed, minced and incubated in the same enzyme solution. The cells were collected and culture dishes with 160,000 viable cells per animal were established. One hundred first-division metaphases per animal were examined for CA. Aberrations were classified separately by type (chromatid or chromosome) and analysed as rearrangement or breakage events. The replication indices were estimated from 200 metaphases per animal. The number of metaphases per 1000 consecutive cells was also counted for determination of mitotic indices. No increase in the frequency of aberrations per cell and percent aberrant cells and no toxic effects were induced by the treatment: Aberration rate per cell (including chromatid and chromosome breakage as well as rearrangement events) was from 0.04 to 0.07 in treated animals vs. 0.09 in the control group. The few aberrations found were mostly chromatid-type breaks or fragments. Percentages of cells with abnormal chromosomes were not significantly different among treated and control groups within an experiment (6.9 % vs. 6.2 % (for 4000 ppm) and 3.8 % vs. 3.8 % (800 ppm). The replicative and mitotic indices were not significantly different from the controls.
In vivo chromosome aberration, RL2
A mammalian chromosome aberration test was performed with spermatocytes after inhalation exposure in male mice. Methanol was vaporised with a air flow rate of 10^5 L/min and a air change rate of 15 air changes/hours. Animals were treated for 6 hours/day for 5 days with a post-exposure period of 5 days. Doses were 1.04 or 5.3 mg/L air (corresponding to 800 and 4000 ppm). Spermatocytes of the pachytene substage of the meiotic prophase after 5 days of last exposure were isolated, and fixed for electronmicroscopy: Several morphological parameters concerning the meiotic chromosome structure were examined (synaptonemal complex analysis). The so-called "synaptonemal complex (SC)" is the attachment site of homologues chromosomes during meiotic prophase. SC damage is an endpoint of meiotic chromosome aberrations. An increase in the frequency of synaptic lesions may be an indicator for exposure to mutagens. Methanol did not produce significant dose-dependent increases in SC aberrations.
In vivo DNA damage and/or repair, RL2
An in vivo DNA damage study was performed to determine whether MeOH could indirectly DNA damage via ROS-mediated mechanism. Animals (mice, rabbits and monkeys) were treated either once or for 15 days with 2.0 g/kg MeOH i.p. following a post exposure period of 6 hours or 15 days. KBrO3 was used as positive control. Oxidative DNA damage was observed by measuring 8-oxo-2'-deoxyguanosine. Lipid peroxidation in bone marrow and spleen homogenates was determined by measuring the Ievels of HNE-His protein adducts. Three rabbits and primates were used in each treatment group. Four and five mice were used in each group for the acute and chronic studies, respectively. KBrO3 was used as positive control. No increase in oxidative DNA damage was observed in bone marrow or spleen at 6 h following exposure to MeOH (2.0g/kg bw ip) in mice, rabbits, or primates. Similarly, no increase in oxidative DNA damage was observed in bone marrow or spleen 24 h following exposure to a single dose of MeOH (2.0g/kg bw ip), or following 15 consecutive daily doses of 2.0g/kg bw ip MeOH in CD-1 mice. No increase in HNE-His protein adducts was observed in bone marrow or spleen at 6 h following exposure to MeOH (2.0 g/kg bw ip) in primates. MeOH exposure (2.0 g/kg bw ip) increased HNE-His protein adducts 1.4-fold 6 h post-dose in bone marrow of mice, with adduct Ievels returning to basal values within 24 h. No increases in HNE-His protein adducts were observed in spleen of mice or bone marrow of rabbits following MeOH exposure (2.0 g/kg bw ip). MeOH exposure (2.0 g/kg bw ip) increased HNE-His protein adducts 1.5-fold 6 h post-dose in spleen of rabbits. Taken together, these observations suggest that it is unlikely that exposure to MeOH would initiate Iymphomas, particularly in humans, via formation of mutagenic oxidative DNA lesions.
In vivo erythrocyte micronucleus test, RL2
An in vivo erythrocyte micronucleus test was performed similar to OECD guideline 474 in bone marrow cells of male and female mice after single intraperitoneal administration and 30 hours post exposure period. Doses of 1920, 3200 and 4480 mg/kg bw and 2 animals per sex per dose were used. 1000 polychromatic erythrocytes per mouse were scored. There was no significant effect on the frequency of micronuclei in treated as compared to control animals: 1.3/1000 (control and low dose), 2.0/1000 (2nd dose), and 3.7/1000 (top dose). Therefore, methanol was found to be not clastogenic under the test conditions used.
In vivo erythrocyte micronucleus test, RL2
An in vivo erythrocyte micronucleus test was performed similar to OECD guideline 474 in male mice after intraperitoneal administration for 4 days and a post exposure period of 24 hours. Doses of 300, 600, 1200 and 2500 mg/kg bw and 10 animals per dose were used. Caffeine and urethane were used as positive controls. Erythrocytes from blood samples were analysed for possible effects of methanol as function of folate status. Folate deficiency is known to increase the spontaneous incidence of micronucleated erythrocytes and strongly enhance the incidence of micronuclei induced by certain chemicals, and because methanol oxidation via formate to CO2 depends on tetrahydrofolate. Mice were made folate-deficient by maintaining on a diet with no folic acid and 1 % succinyl sulfathiazole, inhibitor of the catalase, and another group received folate-supplemented diet containing 5 mg/kg folic acid. Blood samples were taken at 24 hours after the last dosing. Folate limitation resulted in an about 15-20 times lower folate blood level than in the high-folate groups. No difference in micronucleus frequency (MN in PCEs) and in RNA-positive blood erythrocytes was seen between treated groups and controls while animals treated with the positive control substances responded as expected. The results indicate that methanol is non-clastogenic to the developing erythroblast in bone marrow and does not inhibit red blood cell production in either normal or folate-deficient mice.
In vivo erythrocyte micronucleus test, RL2
An in vivo erythrocyte micronucleus test was performed in female mice after oral administration from gestation day 6 to 10 during pregnancy (treatment: twice daily). The study was performed during a developmental investigation under folate-deficient and -sufficient conditions. Animals were fed an amino acid-based, folic acid-free diet supplemented with either 400 (marginal) or 1200 (normal) nmol folic acid/kg diet and 1% succinylsulfathiazole for 5 weeks prior to mating and throughout breeding and gestation. After oral methanol application, the frequency of MN was examined in maternal peripheral blood and fetal blood. Maternal peripheral blood was collected after a post exposure period of 48 hours and fetal blood was taken at gestation day 18. A dose of 2500 mg/kg bw was used in 17 dams. Vein blood was collected on precleaned microscope slides. Slides were air dried and fixed in absolute methanol on the same day. Fixed slides were stained with 0.1 % acridine orange for 5 min, followed by a 17 min rinse in Sorensen´s M/15 phosphate buffer, pH 6.8. Slides were then wet mounted with cover slips and examined by fluorescent microscopy using a mercury lamp. The frequency of MN was scored in 1000 reticulocytes. The frequency of MN in maternal and fetal reticulocytes was similar among the groups. The MN rate was not associated with either maternal or fetal hepatic folate concentrations. There was no significant difference in the frequency of MN in reticulocytes of fetuses that were affected by malformations compared to fetuses without malformations.
In vivo erythrocyte micronucleus test, RL2
An in vivo erythrocyte micronucleus test was performed similar to OECD guideline 474 in male mice after inhalative administration for 5 days (6 hours/day). Doses of 1.04 and 5.3 mg/L (corresponding to 800 or 4000 ppm) were used. 5 animals per dose were treated. Peripheral blood cells and primary cultures of lung cells were analysed. Immediately following the last exposure, animals were anesthetised and blood smears were made from tail vein blood for erythrocyte MN determination. The cells were fixed in methanol and stained with acridine orange for fluorescence microscopy. All slides were coded prior to scoring of 2000 polychromatic erythrocytes (PCE) and 2000 normochromatic erythrocytes (NCE) per animal. The same exposure groups of mice used for blood MN analysis were also used for lung cell MN analysis. After blood was removed by perfusion, the lungs were infused with a trypsin, EDTA and collagenase solution; and then removed, minced and incubated in the same enzyme solution. The cells were collected and culture dishes with 160,000 viable cells per animal were established. Lung MN were analysed in 1000 binucleated cells typically examined from each of 5 animals per dose. Percentages of mononucleated, binucleated, trinucleated and quadrinucleatexd cells were also determined. No treatment related effect on micronuclei frequency or cell kinetics and no toxicity were seen at any dose level.
The available in vivo assays are negative for clastogenicity. Some of these assays were conducted under specific stress conditions using folate-deficient mice (Am. Petrol. Inst., 1991; Fu, 1996).
Conclusion
In conclusion, the majority of in vitro assays are negative for mutagenicity. Positive results from in vitro clastogenicity results are disproved by vivo cytogenicity results which showed no clastogenicity. In summary, methanol is not considered genotoxic.
Overall conclusion:
Based on the in vitro and in vivo genetic toxicity results of the two individual constituents, the reaction mass of methyl acetate and methanol is not classified for genetic toxicity.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The reaction mass of methyl acetate and methanol is assessed on the basis of a read-across approach with the individual constituents methanol and methyl acetate. The available test data for the individual substances are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the reaction mass of methyl acetate and methanol is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.
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