Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Methanol has been examined in numerous tests including bacterial, mammalian and fungal test systems. Most studies produced consistently negative results, with four exceptions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro, other
Remarks:
Type of genotoxicity: genome mutation in fungi
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:
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
Key result
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
Additional information on results:
For the phrase selection in field 'Endpoint' the following source information was used as basis:
Type of genotoxicity: genome mutation
Type of study: other: Mitotic recombination in ascomycetes
Guideline:
Species/strain (Method):
- other: Aspergillus nidulans diploid strain P1

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 %.

Conclusions:
Interpretation of results:
positive
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:
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:
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.
Key result
Species / strain:
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
Key result
Species / strain:
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:
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:
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
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
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

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).

Conclusions:
Interpretation of results:
ambiguous
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:
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.
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
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
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

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.

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:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
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
Key result
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
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: mammalian cell gene mutation assay
Guideline:
Species/strain (Method):
- mouse lymphoma L5178Y cells
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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.

Conclusions:
Interpretation of results:
positive
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

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

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:
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:
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

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)

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:
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:
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, 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
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
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
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

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:
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:
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, 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
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
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
Conclusions:
Interpretation of results:
negative
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The available in vivo assays are negative for mutagenicity and clastogenicity.

Link to relevant study records

Referenceopen allclose all

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:
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
Remarks:
Doses / Concentrations:
1920; 3200; 4480 mg/kg
Basis:
nominal conc.
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

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).

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
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 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
Remarks:
Doses / Concentrations:
2 g/kg bw
Basis:
nominal conc.
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(s): is a known renal carcinogen
- Route of administration: i.p.
- Doses / concentrations: 100 mg/kg bw at a fixed volume of 0.1 mL/10g 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 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:
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 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
Remarks:
Doses / Concentrations:
1.04 or 5.3 mg/L (corresponding to 800 or 4000 ppm)
Basis:
nominal conc.
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 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:
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 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
Remarks:
Doses / Concentrations:
1.04 or 5.3 mg/L (corresponding to 800 or 4000 ppm)
Basis:
nominal conc.
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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro 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., 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 studies:

The available in vivo assays 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).

In conclusion, the majority of in vitro and in vivo assays on methanol are negative for mutagenicity and clastogenicity. However, a few of the in vitro assays were positive or ambiguous (DeFlora et al., 1984a, 1984b; McGregor et al., 1985; Crebelli et al., 1989). The positive findings can not be evaluated since the available data base is limited.


Short description of key information:
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium negative except TA 102+S9 (ambiguous) (OECD 471)
Gene mutation (Mammalian cell gene mutation assay): V79 negative, L5178Y+S9 positive (both comparable to OECD 476)
Chromosome aberration (in vitro micronucleaus assay): V79 negative
DNA damage (Damage and repair assay in bacteria): E. coli positive
Genome mutation (Mitotic chromosomal segregation assay): A. nidullans positive

In vivo:
Chromosome aberration (Chromosomal aberration): primary lung cells negative
Chromosome aberration (Micronucleus assay): erythrocytes negative (similar OECD 474), primary lung cells negative
Chromosome aberration (Synaptonemal complex): pachytene spermatocytes negative

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on th negative results in the in vivo studies, methanol does not appear to be mutagenic. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.