Dodecanoic acid, 3-[[2-(2-hydroxyethoxy)ethyl]imino]-2,2-dimethylpropyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sika Hardener LG was examined in one in vitro genetic toxicity study using a bacterial reverse mutation assay (Ames test). Results showed that Sika Hardener LG did not induce gene mutations by frameshift or base-pair substitution in the examined strains tested up to cytotoxic concentrations. Thus, Sika Hardener LG was considered non-genotoxic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-08-29 to 2008-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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 / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbitone/beta-naphtoflavone-induced rat liver S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium :10%
- quality controls of S9: Biological activity of each batch of S9 was characterised in the Salmonella assay (using 2-Aminoanthracene and Benzo(a)pyrene. which require metabolic activation by microsomal enzymes). Sterility test was performed (on blood agar).

Test concentrations with justification for top dose:

Based on the results of preliminary tests the highest dose was 5000 pg test item/plate in the final treatment mixture under the actual conditions of the test at the start of the experiment.
Mutation Test and Confirmatory Mutation Test the tested concentrations were: 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate. This series was completed in the Confirmatory Mutation Test with additional concentration levels of 5, 1.581, 0.5,0.1581 and 0.05 µg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For the S. typhimurium TA 1535 strain, without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For the S. typhimurium TA 1537 strain, without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For the E. coli WP2 uvrA strain, without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : three

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 °C
- Exposure duration/duration of treatment: at 37 °C for at least 48 hours in the dark

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
In the Initial Mutation Test (Experiment I.), Confirmatory Mutation Test and Complementary Confirmatory Mutation Test the observed revertant colony number increases with mostly minor intensity, not dose related, below the biological relevant threshold value and in the historical control range, so they were considered as reflecting the biological variability of the test. In the Initial Mutation Test the highest revertant rates were observed in the in case of Salmonella typhimurium TA1537 test strain at 500, 158.1 and
50 µg/plate plate concentration (mutation factor values of 1.93, 1.93 and 2.14 , respectively) with metabolic activation (+S9 Mix). However, the revertant colony numbers remained in the historical control range, there was no dose dependence and these values are below the biological relevant threshold value.
Reduced number of revertant colonies were detected in the Initial Mutation Test in Salmonella typhimurium TA98 tester strains with metabolic activation at 5000, 1581 and 500 µg/plate concentration, however the numbers of revertant colonies are comparable to number of revertant colonies grew on the untreated control plates, furthermore the values are in the historical control range.

No revertant growth was observed in the Initial Mutation Test in Salmonella typhimurium TA1537 strain with metabolic activation at 5000 µg/plate concentration, although no difference in the background lawn was detected.
In the Confirmatory Mutation Test (Experiment II) an increase in the number of revertant colonies (compared to the solven control) and higher revertant rates were observed in case of Salmonella typhimurium TA98 test strain at 158.1, 50 and 15.81 µg/plate concentration (mutation factor values of 1.63, 1.55 and 1.45, respectively) with metabolic activation (+S9 Mix). but these MF values are comparable to the data of the untreated control plates (MF value of 1.66) in that experiment.

Lower number of revertant colonies was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain without metabolic activation at 158 1, 500, 158.1, 50 µg/plate concentrations. however those values remained in the historical control range. An inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in the examined Salmonella typ. TA98, TA1535 and TA1537 strains. In case of TA98 and TA1535 without metabolic activation system (-S9 Mix) reduced background lawn development was observed at the concentration of 5000 µg/plate. In case of TA98, TA1535 and TA1537 with metabolic activation (+S9 Mix) reduced background lawn development was observed at 5000, 1581 and 500 µg/plate concentration. In case of TA1537 tester strain without metabolic activation system (-S9 Mix) reduced or slightly reduced background lawn development was observed from 5000 to 50 µg/plate concentration range. In these cases besides the inhibited background lawn development the number of revertant colonies (compared to the revertant colony numbers of the solvent control plates) were also reduced. No revertant growth was observed in TA1535 and TA1537 strains with metabolic
activation at the highest dose of the test item.

Reduced revertant rates (compared to the data of the solvent control plates) were detected in the Complementary Confirmation Mutation Test (Experiment III) in Salmonella typhimurium TA1537 strain without metabolic activation at 15.8 1, 5 and 1.58 1 µg/plate. In this case, the increased revertant colony number of the solvent control (mean value of revertant was 7.3 in contrast of mean value of 3.7 measured in the Confirmatory Mutation Test) caused the reduced revertant rates.

After 48 hours incubation microdrops were observed at the concentration of 5000 µg/plate (with and without S9 Mix), using both the plate incorporation and preincubation method in case of all tester strains.

Conclusions:

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 LG was considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

Sika Hardener LG was assessed in one in vitro genetic toxicity study. The purpose of the 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 LG was considered non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Based on the results of the of the in vitro genetic toxicity study , Sika Hardener LG was not classified as genotoxic according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.