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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
publication
Title:
Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds.
Author:
Wangenheim, J. and Bolcsfoldi, G.
Year:
1988
Bibliographic source:
Mutagen. 3(3):193-205.

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethanol
EC Number:
200-578-6
EC Name:
Ethanol
Cas Number:
64-17-5
Molecular formula:
C2H6O
IUPAC Name:
ethanol
Details on test material:
- Supplier AB Svenska Sprit (Sweden)
- Purity: highest available (not specified)

Method

Target gene:
TK forward mutation
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.