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Genetic toxicity: in vitro

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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:
2008-10-14 until 2008-11-20
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:
2009
Report Date:
2009

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:
2008
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
Details on test material:
- Name of test material (as cited in study report): Aldimine 1
- Physical state: liquid, almost colourless
- Analytical purity: 96.1%
- Lot/batch No.: UB2.398A/07
- 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 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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type 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 Concentrations ranged between 0.001581 µg/plate to 5000 µg/plate.
In experiment I (plate incorporation method) and experiment II (pre-incubation method):
5000; 1581; 500; 158.1; 50, 15.81 µg/plate.
In experiment III (plate incorporation method):
500; 158.1; 50; 15.81 and 5 µg/plate for TA 100 with and without metabolic activation system and for TA 1537 with metabolic activation system.
In experiment IV (pre-incubation method):
50; 15.81; 5; 1.581; 0.5, 0.1581 µg/plate for TA 1535 with and without metabolic activation system
15.81; 5; 1.581; 0.5; 0.1581, 0.05 µg/plate for TA 1537 with and without metabolic activation system.
In experiment V (pre-incubation method):
15.81; 5; 1.581; 0.5; 0.1581; 0.05; 0.01581; 0.005, 0.001581 µg/plate for TA 1537 without metabolic activation system.
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
, water
Negative solvent / vehicle controls:
yes
Remarks:
, acetone, DMSO
True negative controls:
no
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 experimental conditions:
- 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 / choice of top concentrations:
no cytotoxicity
Remarks:
, although a cytotoxic effect of the test item was observed in some strains only in experiment I (using the plate incorporation method) and in experiment II (using the pre-incubation method).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment I (Initial Mutation Test) based on the results of the range finding pre-experiment, the following concentrations were used:
5000; 1581; 500; 158.1; 50 and 15.81 μg/plate.
In the Initial Mutation Test for Salmonella typhimurium TA98 test strain no higher revertant rate was observed than the upper limit of the normal range. An inhibitory effect of the test item was observed at 5000 μg/plate concentration with metabolic activation (MF: 0.14), although the background lawn was not affected.
In case of Salmonella typhimurium TA100 bacterial strain no higher revertant rate was observed than the upper limit of the normal range. Lower number of revertants compared to the control plates were detected at 5000, 500, 158.1, 50 μg/plate without without metabolic activation and at 5000 and 1581 μg/plate with metabolic activation, the obtained number of revertants were below the historical control data range.
For Salmonella typhimurium TA1535 test strain very slightly higher number of revertants (at 500 μg/plate without metabolic activation) and very slightly lower number of revertants (at 50 μg/plate without metabolic activation) compared to the solvent control were detected, all other mutation factor values remained in the normal range.
In case of Salmonella typhimurium TA1537 bacterial strain the highest number of revertant colonies compared to the control was observed at 1581 and 15.81 μg/plate concentrations without metabolic activation system (MF: 1.83 in both cases). However, the revertant colony numbers remained in the historical control range and there was no dose-dependence.
A strong inhibitory effect was observed by using this test strain at 5000 and 1581 μg/plate concentrations with metabolic activation (no revertant colonies grew on the plates at the highest dose), although no difference in the background lawn was detected.
For Escherichia coli WP2 uvrA test strain sporadically higher number of revertant colonies compared to the control plates were observed (the highest mutation factor value was 1.36 at 15.81 μg/plate without metabolic activation), but there was no dose dependence and the number of revertant remained in the historical control range in all cases. Microdrops were observed at the concentration of 5000 g/plate with and without metabolic activation system (S9 Mix) in the Initial Mutation Test in all tester strains after 48 hours of incubation.

In the complementary Experiment III (Complementary Initial Mutation Test) the additional concentration were investigated based on the results of the Experiment I to complete data. The tested concentrations were 500; 158.1; 50; 15.81 and 5 g/plate for TA100 bacterial strain with and without metabolic activation system and for TA1537 bacterial strain with metabolic activation system.
For TA100 bacterial strain very slightly higher number of revertant colonies compared to the control were detected at some doses with or without metabolic activation (highest mutation factor value was 1.25 at 158.1 µg/plate with metabolic activation), but all of the observed revertant numbers were in the historical control range and there was no dose-dependence.
For TA1537 bacterial strain lower number of revertant colonies compared to the control plates was observed at 500 and 158.1 g/plate concentrations with metabolic activation. However the numbers remained in the historical control range and no difference in the background lawn development was detected, so these data were considered as the biological variability of the test. Because the result of the Initial Mutation Test was negative, in the Confirmatory Mutation Test (Experiment II) the same concentrations of SIKA Hardener LI were tested by using the pre-incubation method.
For Salmonella typhimurium TA98 strain slightly higher revertant rates were observed without metabolic activation in the whole tested concentration range (MF values were in the 1.30-1.59 range), but those values were comparable to the revertant rate of the untreated control plates (MF value of 1.50) and the number of revertant colonies was in the historical control range in all cases.
In case of Salmonella typhimurium TA100 test strain a slightly higher number of revertants compared to the control plates was observed at 1581 μg/plate concentration without metabolic activation, all other values remained in the normal range.
For Salmonella typhimurium TA1535 test strain slightly reduced background lawn development was observed at the concentrations of 5000; 1581; 500 and 158.1 μg/plate without metabolic activation, but the number of revertant colonies were in the historical control range in all cases. Lower number of revertants compared to the control was observed at 5000; 1581; 500 and 158.1 μg/plate concentrations with metabolic activation, but no difference in the background lawn development was detected.
In case of Salmonella typhimurium TA1537 bacterial strain an inhibitory, cytotoxic effect of the test item was observed without metabolic activation at the concentrations of 5000; 1581; 500; 158.1 and 50 μg/plate. Besides the slightly reduced background lawn reduced number of revertant colonies was detected in these cases.
Lower number of revertants compared to the control was observed sporadically by using the metabolic activation system, but no difference in the background lawn development was detected and the number of revertants was in the historical control range.
For Escherichia coli WP2 uvrA test strain sporadically lower number of revertant colonies compared to the control plates was observed with or without metabolic activation, but the number of revertants remained in the historical control range.
Microdrops were observed at the concentration of 5000 µg/plate in all tester strains with and without metabolic activation system after 48 hours incubation in the Confirmatory Mutation Test.

In the complementary Experiment IV (Complementary Confirmatory Mutation Test) additional concentration levels were investigated based on the results of the Experiment II to complete the data. The tested concentration were additional concentrations were 50; 15.81; 5; 1.581; 0.5 and 0.1581 g/plate for TA1535 with and without metabolic activation system and 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate for TA1537 with and without metabolic activation system.
In case of Salmonella typhimurium TA1535 the highest revertant rate was observed at 50 µg/plate with metabolic activation (MF value of 1.35), but the number of revertant colonies was in the historical control range, this mutation factor value is well below the biological relevant threshold value and there was no dose-dependence.
The revertant rates observed at the concentration range of 50-1.581 µg/plate without metabolic activation system were slightly lower than the lower limit of the normal range, but the numbers of revertant colonies were in the historical control range.
For Salmonella typhimurium TA1537 bacterial strain the highest revertant rate was observed at 5 µg/plate with metabolic activation (MF value of 1.57). This mutation factor value is below the biological relevant threshold value, and the number of revertant colonies was in the historical control range. Slighty reduced background lawn and lower revertant rates compared to the control was observed at 15.81 and 5 µg/plate concentration without metabolic activation system. In the complementary Experiment V (2nd Complementary Confirmatory Mutation Test) more additional concentrations were investigated for TA1537 bacterial strain to complete the data and meet the requirements of the OECD Guideline. The tested concentration were 15.81; 5; 1.581; 0.5; 0.1581; 0.05; 0.01581; 0.005 and 0.001581 µg/plate without metabolic activation system For Salmonella typhimurium TA1537 test strain the highest revertant rate was observed at 0.1581 μg/plate concentration (mutation factor value of 1.94). However, the mutation factor value is below the biological relevant threshold, there was no dose-dependence and the number of revertant colonies was in the historical control range. Lower revertant rate compared to the control and slightly reduced background lawn development was observed at 15.81 µg/plate concentration. Positive and negative controls were run concurrently in each experiment. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. In each test the viability of the bacterial cells were checked by a plating experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

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

SIKA Hardener LI is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

SIKA Hardener LI was assessed in an Ames test according to EU method B.13/14, OECD guideline 471 and EPA OPPTS 870.5100. 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 LI. Five independent experiments were carried out, including two plate incorporation tests (Experiment I, and Experiment III) and in three pre-incubation tests (Experiment II, Confirmatory Mutation Test; Experiment IV, Complementary Confirmation Mutation Test and Experiment V, 2nd Complementary Confirmatory Mutation Test). Experiments I-IV were carried out with and without metabolic activation (S9 Mix), Experiment V was performed without metabolic activation. Concentrations up to 5000μg/plate (limit concentrations) and concurrent controls were tested in triplicates.

Using the plate incorporation method (Experiment I and III) a higher number of revertant colonies was observed compared to solvent controls, but considered to be of minor intensity and not dose related. Mutation factors were below the biological relevant threshold and the number of revertant colonies remained within the historical control range. Therefore, the observations were considered to reflect biological variability.

An inhibitory, cytotoxic effect of the test item was observed in some strains using the plate incorporation method. In Experiment I (Initial Mutation Test) strong reductions in the number of revertant colonies were observed, although no difference in background lawn development was noted. After 48 hours incubation using the plate incorporation method, microdrops were observed in all tester strains at concentrations of 5000 ug/plate, with or without metabolic activation.

Using the pre-incubation method (Experiment II, IV and V) a higher number of revertant colonies was observed compared to solvent controls. However, again the increased number of revertant colonies was of minor intensity and did not follow a dose-response relationship. Mutation factors were below the biological relevant threshold and the number of revertant colonies within the historical control range. Therefore, these observations too were considered to reflect biological variability.

An inhibitory, cytotoxic effect of the test item was observed in some strains using the pre-incubation method. In Experiment II (Confirmatory Mutation Test) slightly reduced background lawn was observed. Further, numbers of revertant colonies were reduced. After 48 hours incubation microdrops were observed in all tester strains at concentrations of 5000 µg/plate with or without metabolic activation.

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. In each experiment the viability of the bacterial cells was checked by a plating experiment.

In conclusion, the data of this mutagenicity assay 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.