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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:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylbenzene
EC Number:
202-849-4
EC Name:
Ethylbenzene
Cas Number:
100-41-4
Molecular formula:
C8H10
IUPAC Name:
ethylbenzene
Details on test material:
- Name of test material (as cited in study report): Ethylbenzene
- Physical state: colorless liquid
- Analytical purity: 99.85%
- Lot/batch No.: 01353MC

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 liver homogenates
Test concentrations with justification for top dose:
Assay A1: 0, 4.2, 8.3, 16.6, 33.1, 66.25, 132.5, 265, 530 and 1060 µg/ml.
Assay B1: 0, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120 µg/ml.
Assay C1: 0, 10, 30, 38, 42, 46, 50, 54, 60, 70 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulfonate and 20-methylcholanthrene
Details on test system and experimental conditions:
no data
Evaluation criteria:
The activation and non-activation assays were considered independent assays with their own negative and positive controls. For an assay to be considered acceptable, (1) the mutation frequency of positive controls should have been significantly higher, 95 x 10-6 above the average of the concurrent solvent controls, and (2) the mutant frequency of the negative controls should have been within 35-140 x 10-6. The negative controls must have had an average absolute cloning efficiency between 65- 120% and a cumulative suspension growth greater than eight.
Mutant frequencies were evaluated based upon biological significance criteria (Moore et al., 2003). The test chemical was considered positive when the conditions listed below were met:
a) the average mutant frequency in at least one dose level of the treated cultures (resulting in ≥ 10% relative total growth) was 95 x 10-6 above the average of the concurrent solvent controls (assuming these to be in the range of 35-140 x 10-6).
b) There was a positive dose related linear trend. This was tested using a linear trend test at alpha = 0.05, provided the above criterion was met.
The test material was considered negative in this assay if the following condition was met:
a) There was no evidence of increase in mutant frequency at RTG values ≥ 10%.
The test material was considered equivocal in this assay if the following conditions were met:
a.) there was a significant increase in mutant frequency that met the criteria for a positive response only at RTG values > 10% and < 20%.
b.) there was no evidence of increase in mutant frequency at RTG values ≥ 20%.

Statistics:
no data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: In absence of S9, the test material was excessively toxic at the five highest concentrations (66.25, 132.5, 265, 530 and 1060 µg/ml.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
There was no appreciable change in either the pH or the osmolality of the culture medium following the addition of the test material as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.56; osmolality = 432 mOsm/kgH2O; culture medium with 1% DMSO, pH = 7.43, osmolality 447 mOsm/kgH2O).

COMPARISON WITH HISTORICAL CONTROL DATA:
Cultures treated with the positive control chemical induced a positive response as compared to the solvent control. The solvent control values were within the range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary toxicity assay (Assay A1), the test material was evaluated at concentrations ranging from 4.2 to 1060 mg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration represented the limit dose of 10 mM and exceeded the solubility of the test material in the treatment medium. In the absence of S9, the test material was excessively toxic at the five highest concentration levels (i.e.,66.25, 132.5, 265, 530 and 1060 mg/ml) as measured by Day 2 relative suspension growth (RSG). The remaining cultures had day 2 RSG values ranging from 52 to 107% (Table 1A). In the presence of S9, excessive toxicity was observed at the 132.5 mg/ml concentration level and above as measured by Day 2 RSG. The remaining concentration levels had RSG values ranging from 15 to 95% (Table 1B). Based upon the results of this assay, concentrations in the range of 10 to 120 mg/ml were selected for the initial mutagenicity assay both in the absence and presence of S9.
Remarks on result:
other: other: The cell line, TK +/- -3.7.2C clonal line of L5178Y mouse lymphoma cells were used in the study
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based upon the frequency of TK-/- mutants in the treated cultures, it was concluded that ethylbenzene was non-mutagenic in the absence and presence of S9 in the assay system employed.
Executive summary:

Ethylbenzene was evaluated in anin vitromouse lymphoma (L5178Y TK+/-) forward mutation assay. The genotoxic potential of the test material was assessed in two independent assays at concentrations ranging from 10 to 120 mg/ml in the absence and presence of an externally supplied metabolic activation (S9) system. The highest concentration tested in an initial toxicity assay (i.e., 1060 mg/ml) was the limit dose of 10 mM and exceeded the solubility of the test material. There were no significant increases in mutant frequency in the test material treated cultures as compared to the solvent controls in the absence or the presence of S9. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, methyl methanesulfonate for assays without S9 and 20-methylcholanthrene for assays with S9. Negative control cultures were treated with the solvent used to dissolve the test material (i.e., dimethyl sulfoxide). Based upon the frequency of TK -/- mutants in the treated cultures, it was concluded that ethylbenzene was non-mutagenic in the mouse lymphoma assay.

 

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