Registration Dossier
Registration Dossier
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EC number: 203-016-8 | CAS number: 102-24-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found in the body fluids and tissues following absorption by any route of administration. Therefore, an assessment of genotoxic potential was conducted taking account of the hydrolysis breakdown products of TMBX.
In vitro gene mutation studies in bacteria (Stewart, 1991) in vitro gene mutation studies in mammalian cells (Rudd, 1991) and in vitro cytogenicity studies (NTP, 1987) concluded that boric acid is not genotoxic under the conditions of the studies.
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., 1984); 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).
Overall from the weight of evidence presented, the in vitro genotoxic potential of TMBX is negative.
Link to relevant study records
- Endpoint:
- genetic toxicity in vitro
- Remarks:
- Type of genotoxicity: genome mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
- Principles of method if other than guideline:
- Determination of chromosomal malsegregation in ascomycetes during mitosis induced by test substance application.
- GLP compliance:
- not specified
- Type of assay:
- other: Mitotic recombination in ascomycetes
- Target gene:
- not applicable
- Species / strain / cell type:
- other: Aspergillus nidulans diploid strain P1
- 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:
- 5.2, 5.6, 6.0, 7.0 % (v/v)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Species / strain:
- other: Aspergillus nidulans diploid strain P1
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 7.0 % (lowest concentration to arrest conidial germination)
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results (migrated information):
positive - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comprehensive test programme using test procedures in accordance with accepted standard methods, but 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 97
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- 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
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results (migrated information):
negative - 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:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well documented with methodological restrictions.
- 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(s)/solvent(s) 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.
- 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
- Positive controls validity:
- not specified
- 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
- 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
- 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
- Positive controls validity:
- not specified
- Remarks on result:
- other: other: microwell method
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
ambiguous - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comprehensive test programme using test procedures in accordance with accepted standard methods, but 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)
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- non-toxic within the range of testing
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- 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
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results (migrated information):
negative - 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:
- - Vehicle(s)/solvent(s) 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.
- 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
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results (migrated information):
negative - Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Only abstract, no detailed data.
- Principles of method if other than guideline:
- Optimisation of the amount of S9-mix to be used to obtain maximum yield of mutations. Comparative study including typical mutagens. According to Clive and Spector (1975), Mutat Res 31: 17-29.
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidin-kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 7.9 mg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Details on test system and experimental conditions:
- DURATION
- Exposure duration: at least 4 h
SELECTION AGENT (mutation assays): TFT (trifluorothymidine) - Evaluation criteria:
- Significant increases in mutation frequency with the test substance
- Statistics:
- no data
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive - 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(s)/solvent(s) 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
Migrated to IUCLID6: 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 (if incubation with a selection agent): after 6 days
SELECTION AGENT (mutation assays): 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.
- 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
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information):
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14-05-91 to 12-08-91
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevent results.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- There is a failure to justify the maximum concentration of 2500 ug/plate
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA 40 CFR Part 158; FIFRA, Section 158.340
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 84-2
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 1538
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9 at 4 % and 10 %
- Test concentrations with justification for top dose:
- 10; 50; 100; 1000; 2500 μg/plate
- Vehicle / solvent:
- Water
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Positive controls:
- yes
- Positive control substance:
- other: 2-Anthramine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In water
DURATION
- Preincubation period: None - Evaluation criteria:
- No data
- Statistics:
- No data
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The study was performed according to Guideline 84-2 and is comparable to OECD 471. The test substance was not mutagenic in any of the strains tested with or without metabolic activation. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- No data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevent results.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Carried out to an internal NTP protocol, which generally complies with OECD guidelines.
- Deviations:
- not applicable
- Principles of method if other than guideline:
- Although this was carried out to an internal NTP protocol, it generally complies with OECD guidelines. Other literature data confirm the results. Also based on Galloway et al; 1985.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix - Aroclor 1254 induced rat (Sprague-Dawley) liver S9 fraction
- Test concentrations with justification for top dose:
- With S-9: 500, 1000, 1500 and 2000 μg/mL.
Without S-9: 1000, 1600, 2000 and 2500 μg. - Vehicle / solvent:
- Water
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In water
NUMBER OF CELLS EVALUATED: Not stated - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Test substance is non genotoxic. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- No data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study.
- Qualifier:
- according to guideline
- Guideline:
- other: 40 CFR Part 158 US-EPA-FIFRA, Section 156.340
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase, TK locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- No data
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix or other (Aroclor 1254 induced rat (Fischer 344) liver S9 fraction used at 1 %).
- Test concentrations with justification for top dose:
- 0, 1.2, 1.7, 2.45, 3.5 and 5.0 mg/mL boric acid.
- Vehicle / solvent:
- No data
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Positive controls:
- yes
- Positive control substance:
- other: Hycanthone methylsulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: ROP plus 5 % heat treated horse serum
NUMBER OF CELLS EVALUATED: Approximately 600/dose - Evaluation criteria:
- Mutations at the TK locus
- Statistics:
- No data
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Concentration related cytoxicity (60 % reduction over controls at 5 mg/mL)
- Additional information on results:
- Concentration related cytotoxicity (60 % reduction over controls at 5 mg/mL).
Increase in ouabain resistance seen (not significant). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance was not mutagenic but cytotoxicity observed at 5 mg/mL (maximum dose level). - 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:
- other: Comprehensive test programme using test procedures in accordance with accepted standard methods, sufficiently documented.
- 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.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- 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
- Positive controls validity:
- not specified
- 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
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results (migrated information):
negative
Referenceopen allclose all
There was a concentration-related increase in non-disjunctions (chromosomal malsegregation) with a maximum of about 3 %. No aneuploidies were noted at the lowest test concentration. The result was statistically significant at two concentrations and a dose-response relationship was evident (IPCS/WHO 1997).
No other forms of malsegregation were statistically significant (1/265 cross-over seen at 6.0 %). The survival was reduced in dose-related manner.
Survival and amount of aneuploidy in Aspergillus nidulans after methanol-treatment:
Concentration [%] |
Survival [%] |
Scored colonies |
Aneuploids (yellow segregants) |
|
counts |
[%] |
|||
5.2 |
26 |
268 |
0 |
0 |
5.6 |
19 |
407 |
6 |
1.47* |
6.0 |
10 |
265 |
8 |
3.02** |
7.0 |
5 |
176 |
0 |
0 |
Spontaneous control |
||||
0 |
100 |
2673 |
7 |
0.26 |
* P<0.01; ** P<0.001
Note: Similar results were obtained with ethanol and other primary aliphatic alcohols. The aneuploidy rate of ethanol reached a maximum of about 10 %.
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.
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).
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
In the presence of 10 - 15 µL/mL S9 and methanol (7.9 mg/mL), there was a significant increase in mutation frequency. No detailed results presented.
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) |
Gene mutation assay results:
Concentration mg/mL |
Number of mutant cells per 106cells ± SD |
Comments give information on cytotoxicity or other |
|||
Exp 1 |
Exp 2 |
Exp 1 |
Exp 2 |
||
-S9 |
-S9 |
+S9 |
+S9 |
||
0 |
54 ± 10 |
42 ± 1 |
29 ± 10 |
36 ± 7 |
|
1.2 |
46 ± 28 |
38 ± 15 |
34 ± 0 |
36 ± 7 |
|
1.7 |
39 ± 17 |
31 ± 9 |
41 ± 7 |
49 ± 4 |
|
2.45 |
27 ± 3 |
32 ± 9 |
40 ± 16 |
36 ± 6 |
Minor cytotoxicity seen |
3.5 |
31 ± 18 |
46 ± 1 |
41 ± 13 |
41 ± 6 |
Cytotoxicity seen |
5 |
50 ± 22 |
41 ± 5 |
53 ± 2 |
47 ± 3 |
Cytotoxicity seen. Increase in + S9 in first study not reproduced. |
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:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found in the body fluids and tissues following absorption by any route of administration. Therefore, an assessment of genotoxic potential was conducted taking account of the hydrolysis breakdown products of TMBX.
The results of an in vivo bone marrow cytogenetic assay (chromosome aberration assay, O’Loughlin 1991) also showed boric acid to be non genotoxic.
The available in vivo assays for methanol genotoxicity are negative for mutagenicity and clastogenicity. Some of these assays were conducted under specific stress conditions using folate-deficient mice (Am. Petrol. Inst., 1991; Fu, 1996). Specifically methanol was found to have no genotoxic potential in erthyrocytes (micronucleus), somatic epithelial lung cells(micronucleus and chromosome aberration) and reproductive tissues (pacytene spermatocytes) (Campell et al 1991)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Based upon the lack of genotoxic potential for the hydrolysis products in in vivo test systems including chromosome aberration, micronucleus formation and germ cell mutation tests, the classificaiton criteria for germ cell mutation set out in 1272/2008/EC are not met.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
