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:
2008-08-29 to 2008-12-18
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1996
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Aldimine 2
- Physical state: liquid, almost colourless / faint yellow
- Analytical purity: 96.7 %
- Lot/batch No.: ub2.398B/11
- Expiration date of the lot/batch: 04 August 2011
- Stability under test conditions: stable for at least 3 years
- Storage condition of test material: Room temperature, closed bottle

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 sys-tem 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
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphtoflavone-induced rat liver S9 mix.
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphtoflavone-induced rat liver S9 mix.
Test concentrations with justification for top dose:
The tests concentrations were: 5000; 1581; 500; 158.1; 50, 15.81,, 5, 1.581, 0.5, 0.1581, 0.05 µg/plate.
Vehicle:
DMSO
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine (NPD)
Remarks:
For the S. typhimurium TA 98 strain, without metabolic activation.
Positive control substance:
sodium azide
Remarks:
For the S. typhimurium TA 1535 strain, without metabolic activation.
Positive control substance:
9-aminoacridine
Remarks:
For the S. typhimurium TA 1537 strain, without metabolic activation.
Positive control substance:
methylmethanesulfonate
Remarks:
For the E. coli WP2 uvrA strain, without metabolic activation.
Positive control substance:
other:
Remarks:
For the S. typhimurium: TA 100; TA 98; TA1535; TA 1537 and E. coli WP2uvrA, with metabolic activation.
Details on test system and conditions:
Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the
mutagenic potential of Sika Hardener LG in three independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in two preincubation tests (experiment II, Confirmatory Mutation Test and experiment III, Complementary Confirmation Mutation Test). Each assay was conducted
with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

- METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension

- The cultures were frozen and were thawed at room temperature. A measured amount was used for inoculating the overnight cultures in the assay.
200 μL inoculum were used for each 50 mL of broth. The bacterial strains were grown in nutrient broth. The cultures were incubated for 10-14 h in a 37°C to the late exponential or early stationary growth phase (approx. 10E9 cells/mL) in a Gyrotory Water Bath Shaker. After the test item was added and mixed and after solidification the plates were inverted and incubated at 37 oC for at least 48 hours in the dark. Revertant colonies were counted manually.

- NUMBER OF REPLICATIONS: 3 replicates per control or concentration levels.
Evaluation criteria:
Evaluation of Experimental Data:
The colony numbers on the control, positive control and the test plates were determined, the mean values and appropriate standard deviations
were calculated. The mutation factor (MF) was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact, not rounded values were used for this calculation).

The test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin,
- the control plates without S9 mix are within the historical control data range,
- corresponding background growth on both negative control and test plates occurs,
- positive controls show a distinct enhancement over the control plate.

A test item is considered as mutagenic if a dose–related increase in the number of revertants occur 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.

A biologically relevant increase is described as follows:
- if in strain TA 100 the number of reversion is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
- if in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.
According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
Mean values and standard deviation were calculated.

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'.
Additional information on results:
In the experiment I. (plate incorporation method) the observed revertant colony number increases compared to the solvent control plates with mostly minor intensity, did not follow a dose-response relationship and were in the historical control range.
Reduced number of revertant colonies were detected in some strains with and without metabolic activation, however values were comparable and remained in the historical control range.
In the experiment II. (pre-incubation method) an increase in the number of revertant colonies compared to the solvent control plates was observed in some strains with and without metabolic activation, but the increases were of minor intensity and comparable to the data of the untreated control plates.
An inhibitory, cytotoxic effect of the test item was observed for some strains with and without metabolic activation. Reduced or slightly reduced background lawn development was observed and the number of revertant colonies (compared to the revertant colony numbers of the solvent control plates) were also reduced.
Lower number of revertant colonies and reduced revertant rates compared to the solvent control plates were observed in some strains with and without metabolic activation, but all of those values remained in the historical control range.
In the experiment III (pre-incubation method) some sporiadic revertant number increases or decreases were observed in some strains with and without metabolic activation, but those increases or decreases were minor intensity and in the historical control range.
After 48 hours incubation microdrops were observed at the concentration of 5000 pglplate with or without metabolic activation, using both the plate incorporation and pre-incubation method in case of all tester strains.
Positive and negative controls were run concurrently. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test, result was acceptable in all cases (including a lower value in the experiment III).
The observed revertant colony number increases did not follow a doseresponse relationship, they were below the biological relevant threshold value and in the historical control range. So in conclusion the results were considered as reflecting the biological variability of the test.

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: non-mutagenic

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 LG is considered non-mutagenic in this bacterial reverse
mutation assay.
Executive summary:

The purpose of this study was to establish the potential of Sika Hardener LG to induce gene mutations in Salmonella typhimurium strains, TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA, using reverse mutation assays (Ames test) by plate incorporation and pre - incubation methods, performed according to EU method B.13/14, OECD guideline no. 471. Three independent experiments were conducted; one plate incorporation tests and two pre – incubation tests. All experiments were carried out with and without metabolic activation (S9 Mix), in concentrations between 0.05 – 5000 µg/plate. Revertant colonies were counted in cultures that were brought to their late exponential or early stationary growth phase and were exposed for 48 hours to the test item. Thereafter the mutation factor (MF) (mean value of revertant counts / mean value of revertant counts of the solvent control) was calculated. During the tests positive and negative controls were run concurrently. The revertant colony numbers of solvent control (DMSO) plates were within the historical control data range. The reference mutagens (4-nitro-1,2-phenylenediamine, sodium azide, 9-aminoacridine, methylmethanesulfonate, 2-aminoanthracene) showed a distinct increase of induced revertant colonies and in each experiment the viability of the bacterial cells was checked by a plating experiment, proving altogether that the test results were valid.

In all tests (pre-incubation or plate incorporation methods), the MF values were below the biological relevant threshold value and the number of revertant colonies remained in the historical control range. When higher number of revertant colonies compared to the revertant colony numbers of the solvent control plates were observed, it was of minor intensity and not dose related, therefore, these few cases were considered as not toxicologically relevant increases and just part of the biological variability of the test.

In conclusion, the mutagenicity assay results show 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 LI was considered non-mutagenic in this bacterial reverse mutation assay.