Triethyl borate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation:

Multiple studies exist with useful data on the potential of ethanol to cause reverse mutation in the commonly used bacterial strains (Ames test) both with and without metabolic activation. Testing is frequently done in association with the use of ethanol as an ‘inert’ vehicle in the testing of other chemicals. Test results are frequently available for tests up to a plate concentration of 10mg/plate. There is no evidence of mutagenicity in the strains TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538).

In a non-standard bacterial forward mutation assay using E coli, ethanol was found to be genotoxic but only at the same concentrations of 15 -20% by volume that were also associated with profound cytotoxicity. Testing was only carried out without metabolic activation. Bearing in mind the massive concentration at which this result was seen and the conjunction with cytotoxicity leads to the conclusion that genotoxicity is not an important characteristic of ethanol. In an assessment of the potential mutagenic potential of ethanol, DNA proficient and deficient strains of E coli bacteria were used in a DNA repair assay. A liquid micromethod procedure to determine the minimum inhibitory concentration was used along with two other techniques (a spot test and treat-and-plate method). Test were carried out both with and without metabolic activation. The liquid micromethod (with and without metabolic activation and the spot test both gave negative results as did the treat-and-plate method without metabolic activation. The reported result with the latter with metabolic activation was equivocal, but there was insufficient information available to judge the significance of this.

Of particular interest as a summary of the genotoxicity of ethanol, the results from using ethanol as a vehicle in a number of guideline reverse bacterial mutation (Ames) assays was reported. Ethanol as a control vehicle, was used in the Salmonells/microsome test at 200 ul/plate in the plate incorporation assay and up to 100 ul/plate in the pre-incubation assay, the latter being equivalent to 79 mg/plate, or approximately 16 times the normal maximum recommended dose level of 5 mg/plate used in regulatory mutagenicity tests. In 1998 it was used as the vehicle for 18 test materials. The mean, minimum and maximum frequencies of revertant colonies for the ethanol vehicle control plates were all comparable to the 1998 vehicle control history profile (320 -340 assays) for all vehicle controls used at this testing laboratory. 

Overall, it can be concluded with confidence that ethanol is not mutagenic to bacteria.

Clastogenicity

The clastogenic potential of ethanol has been examined in a number of assays and using a number of different mammalian cell lines, a number of which are reported in this dossier.  Concentrations tested are usually very high (eg 1-5%), well in excess of those normally recommended and results are generally negative.  Where positive results are reported, these are close to or exceed the normal maximum recommended concentrations for guideline studies. Many studies, typical of most, are usually incomplete because metabolic activation is not used.

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in a number of guideline clastogenicity (chromosome abberation) in vivo assays were reported for two cell lines (human lymphocytes and Chinese hamster lung). As a control vehicle, ethanol was used in the guideline tests at doses of 1%, well in excess of the maximum dose normally recommended for use. In 1998 across the two cell lines, it was used as the vehicle for 19 test materials without metabolic activation and 11 with. The mean, minimum and maximum frequencies of revertant colonies for the ethanol vehicle control plates were all comparable to the 1997 vehicle control history profile (145 assays) for all vehicle controls used at this laboratory during this year.

From the evidence, it can be concluded that there is little evidence for the clastogenicity of ethanol in vitro.

Mammalian cell mutation

Two similar cell mutation studies using mouse lymphoma lymphoma cells in the TK forward mutation assay reported similar results; ethanol was found to be non mutagenic with and without metabolic activation at very high doses up to and including those that cause significant cytotoxicity (typically in the region 0.3 -0.6M.  In a mammalian cell mutation study using S49 mouse lymphoma lymphoma cells that monitored simultaneously selection for ouabain, 6-TG and dexamethasone resistance, ethanol was found to be non mutagenic with activation at a dose of 0.17M.

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in an in vitro guideline mammalian gene mutation assay was reported. As a control vehicle, ethanol was used in the guideline tests at a dose of 1%, well in excess of the maximum dose normally recommended for use. The mean, minimum and maximum mutation frequencies for the ethanol vehicle control plates were all comparable to the pooled results from 20 previous vehicle controls used.

The results from guideline gene mutation assays with ethanol are universally negative. In a study using the more recently developed CBMN (Cytokinesis Blocked Micronucleus) Assay - yet to become guideline, ethanol in the absence of metabolic activation, was found to cause a statistically significant increase in numerical chromosome alterations (aneuploidy) at concentrations above 3mg/ml or 0.067M). In contrast, when the metabolite acetaldehyde was tested, it cause chromosomal alterations (clastogenicity) at concentrations of <0.04mg/ml. It should be noted that the maximum recommended dose for testing chemicals in current standard in vitro protocols is 5mg/ml or 0.01M, which this study clearly exceeded in a number of the tested doses of ethanol.

Link to relevant study records

Reference

Reference 1

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:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, study with sufficient detailed documentation to demonstrate that study meets basic scientific principles and contains enough detail to be able to judge the results reliable as a contribution to the understanding of the genotoxic potential of this substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

, no significant deviations noted

Principles of method if other than guideline:

Method: other: Clive et al. (Muta Res, 59, 61, 1979) with some minor modifications to reduce experiment time and plating efficiency. Study examined a large number of chemicals.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK forward mutation

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher's medium with 10% horse serum, adjusted to pH 7.2
- Source: Boroughs Welcome Co, Research Triangle Park, NC, USA
- Periodically "cleansed" against high spontaneous background: yes by treatement with thymidine, hypoxanthine, methotrexate and glycine

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from Sprague-Dawley rat livers, animals pre-treated with Aroclor 1254.

Test concentrations with justification for top dose:

0.092, 0.184, 0.369, 0.553, 0.738 mol/l without activation; 0.414, 0.465 and 0.517 mol/l with activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive control substance:

cyclophosphamide

Positive control substance:

4-nitroquinoline-N-oxide

Positive control substance:

2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 3 per dose level but 6 for negative control.

DETERMINATION OF CYTOTOXICITY
- Mitotic index: Not strictly applicable. Total growth cf. controls were 88, 84, 53, 34 and 17% from lowest to highest concentrations in the absence of activation. With activation, total growth was 43, 24, and 6% from lowest to highest concentration.

Evaluation criteria:

Two fold or greater increase in mutation frequency at 10% or greater total growth cf. controls.

Statistics:

Tested for normal distribution and then analysis of variance and 2-tailed Student's t-test against controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- None

ADDITIONAL INFORMATION ON CYTOTOXICITY: see table below
- Frequency of reversions etc: Without activation, mutation index values from lowest to highest dose were 1.3, 1.1, 1.2, 1.1 and 1.6. With metabolic activation these values were 1.1, 1.3 and 1.8.
- Dose-effect related observations: No dose-effect related observations were seen.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Only at the maximum concentration, with metabolic activation was total growth <10% control. Without activation, the lowest and highest concentrations of ethanol produced statistically significant increases in mutation frequency.

Without metabolic activation

Concentration (mols/litre)	Total growth	Mutation frequency	Mutation index
0	100%	80, 99	1.0
0.0922	88%	118*	1.3
0.184	84%	94	1.1
0.369	53%	104	1.2
0.553	34%	101	1.1
0.738	17%	140**	1.6

With metabolic activation

Concentration (mols/litre)	Total growth	Mutation frequency	Mutation index
0	100%	63, 46	1.0
0.414	43%	62	1.
0.465	24%	70	1.3
0.517	6%	97**	1.8

*significant, ** highly significant

Rates of spontaneous mutation frequencies in study: without metabolic activation 76 +/-25, with metabolic activation 86 +/-33.

Results are supported by those of Amacher, D., et al. (1980) Mutat. Res. 72:447 -474

Conclusions:

Interpretation of results (migrated information):
negative

Ethanol is judged not to have significant mutagenic activity in this system.

Executive summary:

In a mammalian cell mutation study using mouse lymphoma lymphoma cells in the TK forward mutation assay, ethanol was found to be non mutagenic with and without metabolic activation at very high doses up to and including those that cause significant cytotoxicity (typically in the region 0.3 -0.5M.

Reference 2

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:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published study but containing sufficient details to be able to judge it reliable for hazard assessment. Part of a programme of work by the NTP. Data tables available for results. TA102 not included.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

, no significant deviations noted

Principles of method if other than guideline:

Preincubation as per method of Haworth (Env Mutagen, 5 suppl 1, 3-142, 1983)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture prepared at 37C overnight with shaking in Oxoid #2 broth or in defined minimal medium supplemented with biotin and histidine. Phenotype analysed at time of use.

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture prepared at 37C overnight with shaking in Oxoid #2 broth or in defined minimal medium supplemented with biotin and histidine. Phenotype analysed at time of use.

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture prepared at 37C overnight with shaking in Oxoid #2 broth or in defined minimal medium supplemented with biotin and histidine. Phenotype analysed at time of use.

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture prepared at 37C overnight with shaking in Oxoid #2 broth or in defined minimal medium supplemented with biotin and histidine. Phenotype analysed at time of use.

Species / strain / cell type:

S. typhimurium, other: TA104

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture prepared at 37C overnight with shaking in Oxoid #2 broth or in defined minimal medium supplemented with biotin and histidine. Phenotype analysed at time of use.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male SD rat at 10 and 30% and male Syrian hamster liver S9 fraction, at 10 and 30%.

Test concentrations with justification for top dose:

1; (3; 10; 33;)100; 333; 1,000; 3,333; 10,000 microgram/plate. Concentrations in brackets only tested with TA97 using 30% hamster S9.

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, used for all strains with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine, used for TA98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: used for TA97 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: used for TA1535 and T100 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: used for TA104 without metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins at 37C
- Expression time (cells in growth medium): 2 days at 37C

NUMBER OF REPLICATIONS: 5 plus complete repeat of experiment.

DETERMINATION OF CYTOTOXICITY
- Dose range for main test assessed using TA100. Toxic concentrations defined as those producing a decrease in the background number of his+ colonies and/or a clearing in the background lawn. If no toxicity was seen, the maximum dose tested was 10000ug/plate

Evaluation criteria
Only considered non mutagenic if negative in all strains with and without activation and with both S9 extracts at both concentrations

Evaluation criteria:

Combination of magnitude of increase in number of his+ revertants and shape of dose-response curve.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dose-effected related observations: Ethanol at any dose did not produce a 2-fold increase in his+ revertants in the absence or presence of rat or hamster liver extracts.

Conclusions:

Interpretation of results (migrated information):
negative

Ethanol failed to induce reversions in any S. typhimurium tester strain with or without metabolic activation over a wide range of doses up to 10 mg/plate.

Executive summary:

In a reverse mutation assay in bacteria strains TA97, TA98, TA100, TA104 and TA1535 there was no evidence of mutation up to a maximum plate concentration of 10mg/plate with and without metabolic activation systems derived from two species (rat and hamster) used at two different concentrations.

Reference 3

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

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well reported study with sufficient information to judge it as a reliable contribution to the understanding of the mutagenicity of ethanol. Not tested with metablic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK Locus

Species / strain / cell type:

other: Mouse lymphoma L5178Y cells, TK +/-

Details on mammalian cell type (if applicable):

- Periodically checked for phenotypic stability: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

without

Test concentrations with justification for top dose:

Up to 7.79 x 10-1 M (equivalent to ca. 35.9 mg/ml)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Remarks:

ethanol was being tested as a solvent.

True negative controls:

no

Positive controls:

yes

Remarks:

(10 noncarcinogens and 13 putative animal carcinogens were tested)

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Frequency of dosing: once

NUMBER OF REPLICATIONS: 3 plates each and two controls.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: 6x10E5 cells/ml suspension, 5 log range of concentrations used as range finder. Estimated ID50 used as median dose for main study.
- Protocol: 3 hours treatment followed by cell washing. Cell counts at 24 and 48 hours.
- Mutagenicity test protocol: As cytotoxicity test then split with cells resuspended in soft agar cloning medium, with or without trifluorothymidine.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3hr

OTHER: method of analysis: scintillation counting

Evaluation criteria:

Gene mutation at the thymidine kinase (TK) locus in trifluorothymidine-resistant L5178Y mouse lymphoma cells.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

IC10~2%, IC50 ~6% v/v

Vehicle controls validity:

not applicable

Remarks on result:

other: other: Mouse lymphoma L5178Y cells, TK +/-

Remarks:

Migrated from field 'Test system'.

Ethanol results

Concentration (molarity)	Cell survival	Mutants/10E4 survivors
0	100%	0.73
0.173	91%	0.69
0.26	82%	0.77
0.346	81%	0.81
0.433	75%	0.74
0.52	63%	0.92
6.06	52%	0.68
6.93	36%	0.60
7.79	3%	0.72

No increase in mutants at clearly cytoxic concentrations.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Executive summary:

In a mammalian cell mutation study using mouse lymphoma lymphoma cells in the TK forward mutation assay, ethanol was found to be non mutagenic without metabolic activation at very high doses up to and including those that cause significant cytotoxicity (typically in the region 0.4 -0.6M.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Spot testing with confirmatory incorporation testing with 4 positive controls indicates a valid study. However, there is no mention of Good Laboratory Practice.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, only a single concentration tested.

Principles of method if other than guideline:

This was part of a wider study on the mutagenic potential of ingredients used in cosmetics. Ethanol was one of the solvents used and this record reports the testing done on the vehicles before the main study was completed.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9, 50ul/plate

Test concentrations with justification for top dose:

100 microlitre

Vehicle / solvent:

None. Comparison made with distilled water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control in water for TA1535 and TA100 without metabolic activation at 50ug/plate.

Positive control substance:

9-aminoacridine

Remarks:

Positive control in ethanol for TA1537 without metabolic activation at 50ug/plate.

Positive control substance:

other: 4-nitro-o-phenyene diamine

Remarks:

Positive control in DMSO for TA1538 and TA98 without metabolic activation at 50ug/plate.

Positive control substance:

other: 2-aminoantrhacene

Remarks:

Positive control in DMSO for all strains with metabolic activation at 5ug/plate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).

Salmonella/microsomal assays were carried out by making post-mitochondiral preparations from livers of male Sprague-Dawley rats induced with Aroclor 1254. Reversion of all strains by 5 microgram/plate of the promutagen 2-aminoanthracene was included in each assay system.

Evaluation criteria:

Positive if there was a significant and reproducible increase in the number of colonies on the plate compared to controls. Colonies with a diameter of 0.2mm only counted.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks on result:

other: other: Salmonella typhimurium/microsome test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of mutagenicity was observed for ethanol with and without S9 mix.

Executive summary:

In study that examined the potential for mutagenicity of a number of cosmetic product ingredients, ethanol was used as one of the vehicles for the test substances in a bacterial reversion mutation assay (Ames test). Testing of the vehicle controls against distilled water and untreated plates showed no evidence of mutagenicity with and without metabolic activation at a dose of 100ul ethanol per plate in any of the strains tested (TA98, TA100, TA1535, TA1537, TA1538).

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles The study gave the results expected for positive controls and ethanol evaluated as a solvent to a positive control gave negative results with and without S-9 mix. However, there is no mention of Good Laboratory Practice.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, only a single concentration tested.

Principles of method if other than guideline:

Study examined the potential for mutagenicity of the active ingredient of cannabis in the presence of known mutagens. Ethanol solutions were used and the results of the latter are reported here.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9, purchased from Bionetics Laboratories.

Test concentrations with justification for top dose:

100 microlitre

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

the positive substance used in ethanol solution in this study. Only used with TA1537 as the positive control without S9 at 50ug per plate.

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used in DMSO as the positive control for all strains with S9 at 5ug per plate.

Positive control substance:

other: 4-nitro-o-phenylene diamine

Remarks:

Used in DMSO as the positive control for TA1538 and TA98 without S9 at 50ug per plate.

Positive control substance:

sodium azide

Remarks:

Used in DMSO as the positive control for TA1535 and TA100 without S9 at 50ug per plate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

Different concentrations of 9 delta-tetrahydrocannabinol and positive mutagen controls were added in 0.1 ml proportions together with 0.1 ml of a 16 hr nutrient broth culture of the bacterial test strain or 0.1 ml of the culture and 0.5 ml of S-9 mix. These were then poured onto minimal glucose agar plates to form an even layer across the agar.

NUMBER OF REPLICATIONS: Duplicate plates were made for each strain and plates were incubated for 48 hr at 37 degree C. Colonies were counted on a Quebec colony counter. Background lawn and unreverted bacteria were evaluated by microscopy. Appropriate control combinations and growth study plates were prepared.

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium LT2 strains

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of mutagenicity was observed in the absence or presence of S9 mix.

Executive summary:

In study that examined the potential for mutagenicity of the active ingredient of cannabis in the presence of known mutagens ethanol was used as one of the vehicles for the test substances in a bacterial reversion mutation assay (Ames test). Testing of the vehicle controls against distilled water and untreated plates showed no evidence of mutagenicity with and without metabolic activation at a dose of 100ul ethanol per plate in any of the strains tested (TA98, TA100, TA1535, TA1537, TA1538).

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptably documented study which meets basic scientific principles and contains sufficient detail to be able to judge the results reliable as a contribution to the understanding of the mutagenic potential of this substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, different bacterial strains used

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from Aroclor 1254 pre-treated Sprague-Dawley rats.

Test concentrations with justification for top dose:

up to 160mg/plate

Vehicle / solvent:

no data

Untreated negative controls:

yes

Remarks:

other compounds tested in assay

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium dichromate, acridine derivative ICR 191, phenylhydrazine, hydrogen peroxide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (as derived in Maron and Ames - 1983. Muta Res 113, 1-73).

Evaluation criteria:

Mutagenic potential expressed by dividing number of revertants in excess of controls by corresponding amount of compound in moles.

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Ethanol was negative for mutagenic activity in the Ames reversion test using strain TA97 but showed a reproducible increase in revertants over controls in TA102 but this was less than two-fold increase which is not normally considered to be biologically significant in the Ames test. It was however reproducible. It is unclear if the result was obtained with or without metabolic activation (or with both.) The mutagenic potential (based on most severe response seen with or without metabolic activation in either strain) was 0.00005 revertants/nmole, which can be considered very low.

The authors tentatively classified it as an uncertain or questionable mutagen. However, considering the response against the high dose used (160 mg/plate) suggests that this is an excessively conservative conclusion and that the balance of evidence points to a negative result.

Conclusions:

Interpretation of results (migrated information):
ambiguous

Negative in strain TA97, ambiguous in strain TA102

Executive summary:

As part of a study of the genotoxic activity and potency of 135 compounds, ethanol was evaluated in a bacterial reverse mutation (Ames) assay using the plate incorporation method and both in the presence and absenceof metabolic activation. Strains tested were TA97 and TA102. There was no evidence of mutagenicity in the TA97 strain. A reproducible increase in revertants over controls was seen in TA102 but this was less than two-fold increase which is not normally considered to be biologically significant in the Ames test. It was unclear if this result was obtained with or without metabolic activation (or with both.). The mutagenic potential (based on most severe response seen with or without metabolic activation in either strain) was 0.00005 revertants/nmole, which can be considered very low. The authors tentatively classified ethanol as an uncertain or questionable mutagen based on this result. However, considering the response against the high dose used (160 mg/plate) suggests that this is an excessively conservative conclusion and that the balance of evidence points to a negative rather than an ambiguous result.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles. Consistency of results in the two tests for 71% of the substances tested together with an overall predictive accuracy of 64.5% for the reversion test in 75 compounds classified for their carcinogenic activity, demonstrated the validity of this study on comparability of methods. This study is considered to be valid with restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contained 10% liver S9 fractions from Sprague-Dawley rats pre-treated with Aroclor 1254.

Test concentrations with justification for top dose:

Tested to "toxicity limit" (not defined).

Untreated negative controls:

no

Remarks:

many other substances evaluated in same study.

Negative solvent / vehicle controls:

no

Remarks:

many other substances evaluated in same study.

True negative controls:

no

Remarks:

many other substances evaluated in same study.

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Ethanol tested in revised plate incorporation test as described in Maron, D.M. & Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res. 113; 173-215.

Evaluation criteria:

Mutagenicity potency was expressed by dividing the number of revertants in excess of controls by the corresponding amount of ethanol in moles. A ratio of more than 2 between the MIC's in repair proficient (rep+) and -deficient (rep-) strains was considered to be sufficient.

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 determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

All strains of Salmonells typhimurium showed no significant change in the number of reversions in the presence of ethanol with or without metabolic activation. The potency was (revertants/nmole) < 0.00006.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

As part of a study of the genotoxic activity and potency of 135 compounds, ethanol was evaluated in a bacterial reverse mutation (Ames) assay using the plate incorporation method and both in the presence and absenceof metabolic activation. Strains tested were TA98, TA100, TA1535, TA1537 and TA1538. There was no evidence of mutagenicity in any of the complete battery of strains tested.

Reference 8

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

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles. Consistency of results in the two tests for 71% of the substances tested together with an overall predictive accuracy of 72.4% for the DNA-repair test in 75 compounds classified for their carcinogenic activity, demonstrated the validity of this study on comparability of methods. TRhis study is considered to be valid with restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5500 - Bacterial DNA Damage or Repair Tests

Deviations:

yes

Remarks:

, different bacterial strains used

Principles of method if other than guideline:

Procedure similar to that of Kada (Report of the International Collaboration Program, progress in Mutation Research, Vol 1, Elsevier, 1980) using B Subtilis in the recombination assay system and McCarrol (Env Mutagen, 3, 429, 1981) using various strains of E Coli.

GLP compliance:

not specified

Type of assay:

other: see principles of method below

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

repair proficient

Species / strain / cell type:

E. coli, other: WP67 (uvrA-, polA) and CM871 (uvrA- recA)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contained 10% liver S9 fractions from Sprague-Dawley rats pre-treated with Aroclor 1254.

Test concentrations with justification for top dose:

Tested to "toxicity limit" (not defined).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Bacterial growht was visually evaluated aft 16 hours at 37C by observing the increase in turbidity or the formation of a pellet of settled cells at the bottom of the test wells. Growth of bacterial colonies on agar plates was checked after an additional 8-24 hours incubation at 37C.

Evaluation criteria:

Mutagenicity potency was expressed by dividing the number of revertants in excess of controls by the corresponding amount of ethanol in moles. A ratio of more than 2 between the MIC's in repair proficient (rep+) and -deficient (rep-) strains was considered to be sufficient.

Species / strain:

E. coli, other: WP2 (repair proficient), WP67 (uvrA-, polA) and CM871 (uvrA- recA)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Ethanol showed less a 2 fold or less difference between the minimum inhibitory concentrations (MIC) of the repair deficient and repair proficient strains which was the criteria for deciding on significance. The MICs varied between 5 -20mg, which can be considered high.

In the DNA repair test there was equivocal activity in the 2 hr preincubation assay in the presence of S9 (negative without) and ethanol was inactive in the absence of S9 and in the spot test.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

In an assessment of the potential mutagenic potential of ethanol, DNA proficient and deficient strains of E coli bacteria were used in a DNA repair assay. A liquid micromethod procedure to determine the minimum inhibitory concentration was used along with two other techniques (a spot test and treat-and-plate method). Test were carried out both with and without metabolic activation. The liquid micromethod (with and without metabolic activation and the spot test both gave negative results as did the treat-and-plate method without metabolic activation. The reported result with the latter with metabolic activation was equivocal, but there was insufficient information available to judge the significance of this.

Reference 9

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

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, study with sufficient detailed documentation to demonstrate that study meets basic scientific principles and contains enough detail to be able to judge the results reliable as a contribution to the understanding of the genotoxic potential of this substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

, different cell line

Principles of method if other than guideline:

The mutagenic action is monitored by simultaneous selection for ouabain, 6-TG and dexamethasone resistance.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mammalian cell line, other: S49 mouse lymphoma

Metabolic activation:

with

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1% (0.17M)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Dulbecco's modified Eagle's medium with 4.5g glucose/l, heat inactivated horse serum and fetal calf serum, Bacto Agar, ICR 191 and ethanol ex Sigma. S49.1 ML cells from P Coffino, San Francisco, originally isolated by Horibata and Harris (1970). Cells grown in stationary medium without antibiotics at 37 degree C in a humidified CO2 incubator. Freshly cloned cultures frozen at -80C. Stock cultures frequently discarded and repaced with thawed frozen ones to prevent build up of mutants.

STAIN: Cells stained with trypan blue for counting.

DETERMINATION OF CYTOTOXICITY
- Method: 4 hour treatment. Cells (6x10E6/dose) centrifuged and re-suspended in medium containing S9. Maximum solvent concentration 1%.

Evaluation criteria:

Criteria for positive result: survival >=40%, factor by which frequency elevated compared to control >3, frequency of 6-TG mutants elevated against controls.

Species / strain:

mammalian cell line, other: see below.

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Mean and standard deviation of control: 104 +/- 9
- Elevation of mutant freq. compared to control: 0
- Mutant frequency: 0
- Surviving fraction: 100%

ADDITIONAL INFORMATION ON CYTOTOXICITY: The dexamethasone resistance marker was induced at the highest frequency and was expressed within 3 days after mutagenesis. Ethanol had no effect on the mutagenesis of this marker.

Remarks on result:

other: other: S49 mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

Executive summary:

In a mammalian cell mutation study using S49 mouse lymphoma lymphoma cells in an assay that monitored simultaneously selection for ouabain, 6-TG and dexamethasone resistance, ethanol was found to be non mutagenic with metabolic activation at a dose of 0.17M.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, study with sufficient basic documentation to demonstrate that study meets basic scientific principles and contains enough detail to be able to judge the results reliable as a supporting study. Results only available in graphical form. No positive control.

Qualifier:

no guideline followed

Principles of method if other than guideline:

RK forward mutation assay.

GLP compliance:

not specified

Type of assay:

bacterial forward mutation assay

Species / strain / cell type:

E. coli, other: Escherichia coli RK+ (replicative killing competent strain CHY832)

Details on mammalian cell type (if applicable):

no further data

Additional strain / cell type characteristics:

other: see method description

Metabolic activation:

without

Test concentrations with justification for top dose:

11 to 23% v/v

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(with and without DMSO at 20%)

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Frequency of Dosing: exposure to ethanol for 10 min before plating.
- No. of metaphases analyzed: Not relevant.

STAIN: The test strain carries a lethal gene (RK+) that is repressed below 39 degree C and derepressed above this temperature. After treatment with ethanol at 30 degree C cells were plated and cultured at 42 degree C to detct RK- mutants.

NUMBER OF REPLICATIONS: 3 per concentration.

Evaluation criteria:

Positive result when mutation index twice that of control.

Species / strain:

E. coli, other: Escherichia coli RK+ (replicative killing competent strain CHY832)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

17% v/v

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- None.

DOSE-EFFECT RELATED OBSERVATIONS
- All ethanol preparations induced RK- mutants with mutation indices of 2 or more. Steep dose-response curves showed threshold at 18-19% v/v. Steep dose-response curves showed threshold at 19-19% v/v.

FREQUENCY OF REVERSIONS ETC
- All preparations gave mutation indices up to 50 at the highest dose tested.

MITOTIC INDEX
-Not relevant

Remarks on result:

other: other: Escherichia coli RK+ (replicative killing competent strain CHY832)

Remarks:

Migrated from field 'Test system'.

The 5 ethanol preparations showed similar dose-response curves for induction of RK- mutants with thresholds of 18-19% v/v. Addition of dimethylsulfoxide lowered the thresholds by around 5% to 13-15%. Cytotoxicity was seen at approximately 17% in the absence of DMSO (cytotoxicity was reproducibly virtually absent at 15% and around 1000x greater at 20%.)

Whilst positive, the massive concentration at which this result was seen can be extrapolated to conclude that the result would be negative at more conventional test concentrations. Mutagenicity also only occured at the same concentrations as cytotoxicity.

The results seen with the different ethanol grades were indistinguishable from each other.

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation

Genotoxicity was only seen at the very large doses associated with cytotoxicity.

Executive summary:

In a non-standard bacterial forward mutation assay using E coli, ethanol was found to be genotoxic but only at the same concentrations of 15 -20% by volume that were also associated with profound cytotoxicity. Testing was only carried out without metabolic activation. Bearing in mind the massive concentration at which this result was seen and the conjunction with cytotoxicity leads to the conclusion that genotoxicity is not an important characteristic of ethanol.

Reference 11

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

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although conducted to a standard published method this paper does not present method detail in full. However, it contains sufficient basic documentation to demonstrate that study meets basic scientific principles and contains enough information to be able to judge the results reliable as a supporting study. The study is therefore considered to be valid with restrictions.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A differential DNA repair assay using derivatives of E coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+.

GLP compliance:

not specified

Type of assay:

other: E coli DNA repair host mediated assay

Species / strain / cell type:

E. coli, other: 343/636 (genotype uvrB+/recA+/lac-) and DNA repair deficient 343/591 (uvrB-/recA-/lac+)

Additional strain / cell type characteristics:

other: see method description above.

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from Aroclor 1254 pretreated male Sprague Dawley rats

Test concentrations with justification for top dose:

Up to 1720 mmol/l

Vehicle / solvent:

Solvent not specified, but since ethanol was a solvent used for other compounds and a high concentration was used, it is likely no solvent was used.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: (without S9 mix)

Positive control substance:

cyclophosphamide

Positive control substance:

benzo(a)pyrene

Positive control substance:

ethylnitrosurea

Positive control substance:

methylmethanesulfonate

Positive control substance:

4-nitroquinoline-N-oxide

Positive control substance:

9-aminoacridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: For each concentration of test compound 100 ul of test compound or the solvent, 100 ul of bacterial mix and 500 ul S9 mix (where used) were made up to 1 ml with buffered saline. The mixture was incubated at 37 degree C in the dark before seeding NR agar plates.

Evaluation criteria:

The relative survival of DNA repair deficient and proficient bacteria were calculated.

Statistics:

Confidence interval determined according to the variance of each strain, determined from an experiment with 100 untreated samples. A reduction in number of colonies by 2 standard deviations taken as significant.

Species / strain:

E. coli, other: 343/636 (genotype uvrB+/recA+/lac-) and DNA repair deficient 343/591 (uvrB-/recA-/lac+)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >1720 mmol/l

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

other: Of the 7 positive controls used, 5 gave a positive response, 2 gave a negative response.

Additional information on results:

Note that the positive controls may not necessarily be true positive controls for this DNA repair assay.

Remarks on result:

other: other: 343/636 (genotype uvrB+/recA+/lac-) and DNA repair deficient 343/591 (uvrB-/recA-/lac+)

Remarks:

Migrated from field 'Test system'.

The end point of genotoxicity is the preferential killing of the DNA repair deficient strain. The proficient and deficient strains are incubated together with the substance, with our without metabolic activation, before they are plated, incubated and counted.

Conclusions:

Interpretation of results (migrated information):
negative

In both the absence and presence of S9 mix, the high dose of 1720 mmole/l ethanol gave a negative result in DNA repair deficient strain of E. coli. The authors consider this assay to be a good indicator of genotoxicity in vitro and in vivo.

Executive summary:

In a differential DNA repair assay using derivatives of E coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+, ethanol gave a negative response in both the absence and presence of S9 mix up to and including the highest dose tested of 1720 mmole/l.

Reference 12

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: Well reported study but not a standard assay yet in regular use for the assessment of chemicals. Testing at relatively high doses. No positive control.

Principles of method if other than guideline:

in vitro CBMN assay (Cytokinesis Blocked Micronucleus assay) in conjunction with immunofluorescent labeling of kinetochores.

GLP compliance:

no

Type of assay:

other: CBMN (Cytokinesis Blocked Micronucleus) Assay

Species / strain / cell type:

lymphocytes: human lymphoblastoid cell line MCL-5

Additional strain / cell type characteristics:

other: high level of native CYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAs in plasmids.

Metabolic activation:

without

Metabolic activation system:

Main metabolite acetaldehyde was tested separately in study.

Test concentrations with justification for top dose:

0, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0 % v/v (0.016 to 0.35M or 0.8 to 16mg/l)

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (2E5 cells/ml).

DURATION
- Preincubation period: overnight.
- Exposure duration: 22hrs

CYTOKINESIS BLOCKER: Cytochalasin B.
STAIN (for cytogenetic assays): 20% Giemsa (50% of slides. Remainder stored at -20C for indirect immonofluorescence staining of kinetochores.

NUMBER OF REPLICATIONS: none.

NUMBER OF CELLS EVALUATED: 1000 per slide (2000 per dose) for micronucleus analysis. Up to 100 for kinetochore staining where possible.

DETERMINATION OF CYTOTOXICITY
- Method: % apoptosis and % necrosis.

Evaluation criteria:

For binucleated cells:
1- Cells should have two nuclei of approximately equal size.
2- Cells should not contain more than 6 MN.
3- The two nuclei may be attached by a fine nucleoplasmic bridge which is no wider than 1/4th of the nuclear diameter.
4- The two nuclei may touch but ideally should not overlap each other.
5- MN should have a diameter between 1/16 and 1/3 that of the main nuclei.
6- MN should not be linked or connected to the main nuclei.
7- MN may sometimes overlap the boundaries of the main nuclei.
8- MN should have the same staining intensity as the main nuclei but occasionally staining may be more intense.

For Apoptosis: Cells showing chromatin condensation with intact cytoplasmic and nuclear boundaries as well as cells exhibiting nuclear fragmentation into smaller nuclear bodies within an intact cytoplasm/cytoplasmic membrane.

For Necrosis: Cells exhibiting a pale cytoplasm with numerous vacuoles and damaged cytoplasmic membrane with a fairly intact nucleus as well as cells exhibiting a loss of cytoplasm and damaged/irregular nuclear membrane with a partially intact nuclear structure.

Statistics:

Chi squared with Fisher's exact test.

Species / strain:

lymphocytes: human lymphoblastoid cell line MCL-5

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

There was a a dose dependent increase in the mean frequency of binucleated cells containing micronuclei. This increase at the top dose was approximately 5 times higher in comparison to the control and was statistically significant (P < 0.05) from the 0.4% dose upwards. The statistical significance never reach p<0.01. There was a corresponding decrease in the mean percentage of binucleated cells which was greatly reduced at the top three doses tested (0.8%, 1.0% and 2.0% v/v). There was no effect on the level of apoptosis but there was a statistically significant increase in necrotic cells (massive at the top dose.)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
other: positive, but at high concentration

Executive summary:

In a study using the in vitro CBMN (Cytokinesis Blocked Micronucleus) Assay, ethanol in the absence of metabolic activation, was found to cause cause a statistically significant increase in numerical chromosome alterations (aneuploidy) at concentrations above 3mg/ml or 0.067M). In contrast, when the metabolite acetaldehyde was tested, it cause chromosomal alterations (clastogenicity) at concentrations of <0.04mg/ml. It should be noted that the maximum recommended dose for testing chemicals in current standard in vitro protocols is 5mg/ml or 0.01M, which this study clearly exceeds in a number of the tested doses.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles. The methodology of this test is now accepted and repeatedly used as a standard in vitro test for mutagenicity. It is considered robust for detecting environmental carcinogens. This is however only a summary publication that does not included detailed results and therefore can only be regarded as a supporting study. Not all strains normally tested are reported.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, only two bacterial strains tested.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Not specified for this compound but typically 10, 100, 500, 1000 microgram/plate. (10,000 known also to have been tested).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella/microsome

There were < 70 revertants per 10mg/plate tested.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

As part of a study of the mutagenic potential of 300 chemicals, ethanol was evaluated in a bacterial reverse mutation (Ames) assay at a plate concentration of up to 10mg/plate in the presence of metabolic activation. There was no evidence of mutagenicity in either of the two strains examined (TA98, TA100). . It should be noted that only two of the normal four bacterial strains were evaluated, so the study cannot be regarded as complete or definitive.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The publication cited is a review of the genotoxicity of ethanol. Within the review, data is cited from a number of GLP guideline studies that used ethanol as a vehicle. Whilst experimental detail is lacking from the primary publication, the background control data it refers to can be considered highly reliable

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

Data that is referred to in the publication is clearly carried out to guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

100ul/plate in pre-incubation assays and 200ul/plate in plate incorporation assays

Vehicle / solvent:

For comparison, the results from all other vehicle results are compared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation assays recorded

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 determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of results

ETHANOL	TA100 -S9	TA100 +S9	TA98 -S9	TA98 +S9	TA1535 -S9	TA1535 +S9	TA1537 -S9	TA1537 +S9	TA102 -S9	TA102 +S9
# values	19	19	20	20	18	17	18	18	2	2
Mean	118	131	35	37	30	30	13	15	290	304
SD	6	13	4	1	6	3	4	6
ALL VEHICLES
# values	342	339	337	340	341	331	323	325	20	22
Mean	124	125	31	36	26	18	11	14	262	285
SD	16	16	6	6	5	4	3	3	31	32

For the more common strains, the ethanol results represent around 18 -20 assays (replicates)

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in a number of guideline reverse bacterial mutation (Ames) assays was reported. As a control vehicle, was used in the Salmonells/microsome test at 200 ul/plate in the plate incorporation assay and up to 100 ul/plate in the pre-incubation assay, the latter being equivalent to 79 mg/plate, or approximately 16 times the normal maximum recommended dose level of 5 mg/plate used in regulatory mutagenicity tests. In 1998 it was used as the vehicle for 18 test materials. The mean, minimum and maximum frequencies of revertant colonies for the ethanol vehicle control plates were all comparable to the 1998 vehicle control history profile (320 -340 assays) for all vehicle controls used at Safepharm Laboratories in this year.

Reference 15

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Not a directly reported study but information contained within a comprehensive review of the genotoxic potential of ethanol. Sufficient basic information is provided (including numberical results, to judge this as a contribution to understanding of the genotoxic potential of ethanol. Since the information is also a collation information from of a number of studies carried out to both guideline and GLP, it can be considered highly reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Principles of method if other than guideline:

Information from the use of ethanol as a solvent in guideline and GLP assessments of other chemicals.

GLP compliance:

yes

Remarks:

no data

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Dose of 1% (100ul/10ml) used as part of a vehicle solvent.

Vehicle / solvent:

No

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Control data is control vehicle for all studies carried out at the laboratory.

True negative controls:

no

Positive controls:

no

Positive control substance:

not specified

Remarks:

Migrated to IUCLID6: not reported

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

The values obtained for ethanol as a vehicle control are within the range of mutation frequencies found for all other inert vehicles.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results - mutation frequence (x10E-6):

	Ethanol as a vehicle control		All vehicle controls
	-S9	+S9	-S9	+S9
Number of studies	1	1	20	20
Mean	-	-	95.7	106.6
SD	-	-	32.1	25.7
Max	98.9	134.4	186	148.7
Min	79.6	125.6	31.9	60.3

Experiment with ethanol is from two replicates in a single experiment

Conclusions:

Interpretation of results (migrated information):
negative

Ethanol does not show significant mutation potential in a mammalian cell line when used at a dose in excess of those normally recommended in the OECD guideline.

Executive summary:

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in an in vitro guideline mammalian gene mutation assay was reported. As a control vehicle, ethanol was used in the guideline tests at a dose of 1%, well in excess of the maximum dose normally recommended for use. The mean, minimum and maximum mutation frequencies for the ethanol vehicle control plates were all comparable to the pooled results from 20 previous vehicle controls used at Safepharm Laboratories.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

There is no significant evidence that ethanol is a genotoxic hazard according to the criteria applied normally applied for the purposes of classification and labelling, when data that is only applicable to heavy consumption of alcoholic beverages is excluded, the limit doses normally applied in guideline studies are taken into consideration and the fact that confounding due to other toxic effects associated with very high doses are accepted.