Registration Dossier

Administrative data

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:
2011-05-10 to 2011-07-12
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity S2A: “Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals” (1996) and S2B: Guidance on Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals “(1997)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S-9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S-9 mix
Test concentrations with justification for top dose:
15.8, 50, 158, 500, 1581, 5000 µg/plate
Vehicle:
Dimethyl sulfoxide (DMSO)
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
S. typh. TA98, TA100, TA1535, TA1537 and E. coli with metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: NPD - 4-nitro-1,2-phenylene diamine
Remarks:
S. typh. TA98 without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
S. typh. TA100 and TA1535 without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
S. typh. TA1537 without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli without metabolic activation
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
NA

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the Initial Mutation Test significantly higher revertant colony counts than the revertant colony numbers of the vehicle control plates (but within the actual historical control data range) were observed in S. typhimurium TA1535, at the concentration of 5000 μg/plate, with addition of metabolic activation (+S9 Mix). The MR value was 2.35. This high value was unique and was not accompanied by additional dose-relationship.

In the Initial Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates; furthermore the obtained higher revertant counts were above the corresponding historical control data range in the case of S. typhimurium TA1537 at the concentrations of 500, 50 and 15.8 μg/plate, without metabolic activation (-S9 Mix).

Higher revertant counts were obtained, however within the historical control data range in S. typhimurium TA98, at the concentrations of 5000 and 1581 μg/plate (-S9 Mix) and in the whole concentration range (5000-15.8 μg/plate (+S9 Mix), in S. typhimurium TA1535, at the concentrations of 1581, 50 and 15.8 μg/plate (+S9 Mix), moreover in TA1537 at 15.8 μg/plate (+S9 Mix). The higher revertant colony counts remained the threshold for being positive in all cases.

The revertant colony numbers remained in the vehicle control data range, showed no increases or decreases, but were slightly above the corresponding historical control data ranges in S. typhimurium TA100 at the concentrations of 5000, 158 and 50 μg/plate (-S9 Mix) and at 500 and 15.8 μg/plate (+S9 Mix); in TA1537 at the concentration of 5000 μg/plate, without metabolic activation (-S9 Mix).

In the Initial Mutation Test lower revertant colony counts (than the revertant colony counts of the vehicle control) were observed in the corresponding historical control data ranges in S. typhimurium TA1535 in the concentration range of 5000-158 μg/plate and additionally at 15.8 μg/plate, without metabolic activation (-S9 Mix) and in TA1537 at the concentration of 158 μg/plate (+S9 Mix).

The test item concentrations of Sika Hardener MH tested in experiment II were the same as investigated in the experiment I.

In the Confirmatory Mutation Test, following the pre-incubation procedure inhibitory effect of the test item was observed in all examined bacterial strains at the highest concentration level of 5000 μg/plate, with and without metabolic activation (±S9 Mix). The low revertant colony counts were below the corresponding historical control data ranges, additionally reduced or slightly reduced background lawn development was observed at 5000 μg/plate in S. typhimurium TA98, (±S9 Mix), in TA1537 and E. coli WP2 uvrA (-S9 Mix) and in TA100 (+S9 Mix). The lower revertant colony counts (than the revertant colony counts of the vehicle control plates) remained in the historical control data ranges, but the affected background lawn development indicated the inhibitory effect of the test item at 5000 μg/plate in S. typhimurium TA1535 (±S9 Mix), in TA1537 and E. coli WP2 uvrA (+S9 Mix). No revertant growth and reduced background lawn development was obtained in S. typhimurium TA100, at 5000 μg/plate (-S9 Mix).

The revertant colony numbers were slightly higher than the revertant colony numbers of the vehicle control plates in S. typhimurium TA98, at the concentrations of 1581 and 500 μg/plate (±S9 Mix), in TA1535, at 158 μg/plate (-S9 Mix), and in E. coli WP2 uvrA at the concentrations of 1581, 500, 158 and 15.8 μg/plate (+S9 Mix). These revertant colony number increases remained in the corresponding historical control data ranges. In the Confirmatory Mutation Test slightly lower revertant colony counts (compared to the vehicle control) in the historical control data ranges were observed in S. typhimurium TA98 at the concentration of 15.8 μg/plate (-S9 Mix), in TA1535 at 15.8 μg/plate (+S9 Mix), in TA1537 at 1581 μg/plate (-S9 Mix) and at 158 and 15.8 μg/plate (+S9 Mix). These lower revertant colony counts were within the biological variability of the applied test system.

The revertant colony numbers of vehicle control (DMSO) plates with and without S9 Mix were within the corresponding historical control data ranges in both experiments (Initial and Confirmatory Mutation Test)*.

* With exception of the slightly lower revertant colony numbers of the S. typhimurium TA98.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The revertant colony numbers of the untreated and distilled water control plates in the different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Sika Hardener MH at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Sika Hardener MH was considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item SIKA Hardener MH was tested for mutagenic activity in a bacterial reverse mutation assay according to EU Method B13/14/OECD Guideline 471.

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Sika Hardener MH in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Sika Hardener MH at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values and/or revertant colony numbers above the actual historical control data ranges were observed in both independently performed main experiments.

There was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the Confirmatory Mutation Test following the pre-incubation procedure inhibitory effect of the test item was observed in all examined bacterial strains and included the lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were often below the corresponding historical control data ranges) and reduced or slightly reduced background lawn development. No revertant growth was observed in S. typhimurium TA100 at 5000 μg/plate, without metabolic activation (-S9 Mix).

The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Sika Hardener MH was considered non-mutagenic in this bacterial reverse mutation assay.