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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1993-03-03 to 1993-03-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only 4 bacterial strains investigated

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
84/449/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation (S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3

NUMBER OF INDEPENDENT EXPERIMENTS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, background lawn
Evaluation criteria:
A test article is considered as positive if either a dose-related and reproducible increase in the number of revertants or significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose-related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method was available.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000.0 µg/plate in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects occurred in the test groups with and without metabolic activation in both independent experiments in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 mg/plate with and without S9 mix in all strains used.

MUTAGENICITY DATA:
No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any dose level in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher rates with increasing concentrations in the range below the generally acknowledged border of significance.
In the strains TA 1535 and TA 1537 a seemingly dose-dependent increase in revertant colony numbers were observed in experiment I without metabolic activation. In both strains the factor of 3 which is recommended for a mutagenic effect in these two strains could not be reached. However, the second experiment was carried out as a plate incorporation assay to examine whether the effects in the two strains could be reproduced. The results of the independent second experiment showed absolutely no tendency of a mutagenic effect in the above mentioned strains in the absence of metabolic activation. Therefore, the effect obtained in experiment I is considered not to be biologically relevant.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains.
Executive summary:

In a bacterial reverse mutation assay (Ames test) the mutagenic potential of 1,1'-(1,1,2,2-tetramethylethylene)dibenzene was investigated in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA1537 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate. The test was performed according to OECD 471 and Regulation (EC) 84/449/EEC Method B.13/14.

No toxic effects occurred in the test article groups with and without metabolic activation in two independent experiments in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test item at any dose level in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that under the experimental conditions of a reverse mutation assay (Ames test), 1,1'-(1,1,2,2-tetramethylethylene)dibenzene did not induce point mutations by base pair changes or frameshifts in the genome of four Salmonella typhimurium strains. Therefore, 1,1'-(1,1,2,2-tetramethylethylene)dibenzene is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.