N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:
The test item Sika Hardener MI was tested for genetic toxicity in a bacterial reverse mutation assay according to EU Method B13/14 and OECD Guideline 471. The test item Sika Hardener MI showed no mutagenic activity in the bacterial reverse mutation assay.
HPRT test:
The test item Sika Hardener MI was tested for genetic toxicity in an in vitro mammalian cell gene mutation test according to EU Method B17 and OECD Guideline 476. The test item was not mutagenic in this assay.
Chromosome aberration test:
A chromosome aberration test was conducted according to EU Method B10 and OECD Guideline 473. As a result, the test item was not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-05-10 to 2011-07-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

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. 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 system 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

Species / strain / cell type:

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 / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle 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

Untreated negative controls:

yes

Negative solvent / vehicle 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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typh. TA100 and TA1535 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:

S. typh. TA1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle 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

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

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

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

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

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

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

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 5000, 1581, 500, 158 and 15.8 μg/plate, without metabolic activation (-S9 Mix), and at 5000 μg/plate, with addition of metabolic activation (+S9 Mix); furthermore in TA100, at the concentration of 50 μg/plate (+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) at the concentrations of 1581, 158 and 50 μg/plate (+S9 Mix), in TA1535 in the whole concentration range (5000-15.8 μg/plate) (+S9 Mix), in TA1537, at the concentration of 500 μg/plate (+S9 Mix), moreover in E. coli WP2 uvrA at 5000 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, 50 and 15.8 μg/plate (-S9 Mix) and in the concentrations of 5000, 1581, 500, 158 and 15.8 μg/plate (+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 TA98 at the concentration of 15.8 μg/plate (-S9 Mix), in TA1535, in the whole concentration range of 5000-15.8 μg/plate (-S9 Mix), in E. coli WP2 uvrA at the concentrations of 5000 and 500 μg/plate, with addition of metabolic activation (+S9 Mix).

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

In the Confirmatory Mutation Test significantly higher revertant colony counts than the revertant colony numbers of the vehicle control plates (and above the actual historical control data range) were observed in S. typhimurium TA98, at the concentration of 5000 μg/plate, with metabolic activation (+S9 Mix).

Clear dose-related increase was noticed with the increasing concentrations, but the MR value (was 2.63 at 5000 μg/plate (+S9 Mix)) remained below the biological relevant threshold for being positive.

The revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates in S. typhimurium TA98, at the concentration of 500 μg/plate (-S9 Mix), and at the concentrations of 1581, 500, 158 and 50 μg/plate (+S9 Mix), in TA1535 at 5000 μg/plate (-S9 Mix), in TA1537 at the concentrations of 1581 and 158 μg/plate (+S9 Mix) and in E. coli WP2 uvrA at the concentrations of 5000, 1581, 158 and 50 μg/plate (+S9 Mix). These revertant colony number increases remained in the corresponding historical control data ranges.

In the Confirmatory Mutation Test, following the pre-incubation procedure unequivocal inhibitory effect of the test item was observed in S. typhimurium TA98, TA100 and TA1537 bacterial strains at the highest concentration level of 5000 μg/plate, without metabolic activation (-S9 Mix). The low revertant colony counts were below the corresponding historical control data ranges, additionally slightly reduced background lawn development was observed at 5000 μg/plate in S. typhimurium TA100 and in TA1537 at 5000 μg/plate (-S9 Mix) and beside the slightly reduced background lawn development no revertant growth was obtained in S. typhimurium TA98 at 5000 μg/plate (-S9 Mix).

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 TA1535 at the concentration of 50 μg/plate (-S9 Mix) and at 1581 and 500 μg/plate (+S9 Mix), in TA1537 at 500 and 15.8 μg/plate (-S9 Mix) and at 500 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 MI 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.

The highest revertant colony number increase was observed in the Confirmatory Mutation Test, in the case of S. typhimurium TA98 at 5000 μg/plate (+S9 Mix). The mutation rate was: 2.63.

In the Confirmatory Mutation Test following the pre-incubation procedure inhibitory effect of the test item was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains, without metabolic activation and included the lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were below the corresponding historical control data ranges) and slightly reduced background lawn development. No revertant growth was observed in S. typhimurium TA98 at 5000 μg/plate, without metabolic activation (-S9 Mix).

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

Executive summary:

The test item Sika Hardener MI was tested for genetic toxicity in a bacterial reverse mutation assay according to EU Method B13/14 and 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 MI 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 (were mostly within the corresponding historical control data ranges, see: Validity of the Study). 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.

Significant, revertant colony number increases were observed in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA98, with addition of metabolic activation (+S9 Mix). The significant increases accompanied with dose-relationship, however did not reach the biological relevant threshold for being positive. No further biologically relevant increases (sporadic changes only) were observed in revertant colony numbers of any of the tester strains following treatment with Sika Hardener MI at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

In the Confirmatory Mutation Test following the pre-incubation procedure inhibitory effect of the test item was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains, without metabolic activation and included the lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were below the corresponding historical control data ranges) and slightly reduced background lawn development. No revertant growth was observed in S. typhimurium TA98 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 MI was considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-08-13 to 2014-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital (PB) and β-naphthoflavone (BNF) induced rat livers

Test concentrations with justification for top dose:

Experiment 1 (3 h treatment, harvest 20 h from beginning of treatment):
without S9-mix: 50, 100, 200, 250 µg/mL
with S9-mix: 25, 50, 100, 200, 400 µg/mL

Experiment 2 (20 h treatment, harvest 20 h and 28 h from beginning of treatment):
without S9-mix: 12.5, 25, 50, 100, 150 µg/mL
with S9-mix: 25, 50, 100, 200, 400 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (experiment 1) and 20 hours (experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 400 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was scored.
- Determination of endoreplication: The number of endoreplicated cells was scored.

Evaluation criteria:

The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.
A test item for which the results do not meet the above criteria is considered as non-mutagenic in this system.

Statistics:

The percentage of cells with structural chromosome aberration(s) was calculated.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at concentrations of 400 µg/mL (with S9 mix) in both experiments and at 150 µg/mL without S9 mix in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Mean percentage of cells with structural chromosome aberrations.

Table 1: Experiment A, treatment time 3 h, harvesting time 20 h

Concentration [µg/mL]	S9 mix	Mean aberrant cells / 200 cells including gaps	Mean aberrant cells / 200 cells excluding gaps
Solvent control (DMSO)	-	6	3
50	-	8	3
100	-	8	3
200	-	7	3
250	-	7	4
Ethyl methanesulfonate [1 µL/mL]	-	49**	40**
Solvent control (DMSO)	+	6	3
25	+	7	3
50	+	6	2
100	+	7	3
200	+	9	4
Cyclophosphamide [5 µg/mL]	+	70**	64**

**: p < 0.01

Table 2: Experiment B, treatment time 20 h, harvesting time 20 h

Concentration [µg/mL]	S9 mix	Mean aberrant cells / 200 cells including gaps	Mean aberrant cells / 200 cells excluding gaps
Solvent control (DMSO)	-	7	4
12.5	-	7	3
25	-	8	4
50	-	6	3
100	-	8	3
Ethyl methanesulfonate [0.4 µL/mL]	-	64**	56**
Solvent control (DMSO)	-	7	3
12.5	-	7	3
25	-	7	3
50	-	8	4
100	-	7	3
Ethyl methanesulfonate [0.4 µL/mL]	-	66**	59**

**: p < 0.01

Table 3: Experiment B, treatment time 3 h, harvesting time 28 h

Concentration [µg/mL]	S9 mix	Mean aberrant cells / 200 cells including gaps	Mean aberrant cells / 200 cells excluding gaps
Solvent control (DMSO)	-	6	3
25	+	8	3
50	+	7	3
100	+	9	4
200	+	8	3
Cyclophosphamide [5 µL/mL]	+	68**	53**

**: p < 0.01

Conclusions:

The test substance did not induce structural chromosome aberrations in Chinese Hamster lung cells.

Executive summary:

The test item was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 400 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 50, 100, 200 and 250 μg/mL

with S9 mix: 25, 50, 100, 200 and 400 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 12.5, 25, 50, 100 and 150 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 12.5, 25, 50, 100 and 150 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 25, 50, 100, 200 and 400 μg/mL

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown as the concurrent positive controls, Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations. Based on these results, the substance was determined to not cause chromosomal aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-08-14 to 2014-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat livers

Test concentrations with justification for top dose:

Experiment 1: 5-hour treatment period without S9 mix:
400, 550, 700, 800, 900,1000 and 1100 μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
6.5, 12.5, 25, 50,100, 200 and 400 μg/mL
Experiment 2: 20-hour treatment period without S9 mix:
300, 350, 400, 450, 500, 550 and 600 μg/mL
Experiment 2: 5-hour treatment period with S9 mix:
6.5, 12.5, 25, 50,100, 200 and 400 μg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h and 20 h
- Expression time (cells in growth medium): 8 days
- Fixation time: after selection period

SELECTION AGENT: The selection medium for TG resistant mutants contained 10 μM/mL of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

STAIN: Giemsa

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative survival of the cells treated with 600 µg/mL and above (without S9 mix) and 100 µg/mL (with S9 mix) of the test substance was found to be below 50 % compared to the solvent controls.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1: Mutant frequencies without S9-mix (5 hour treatment)

Concentration [µg/mL]	Mutant Frequency Experiment 1a	Mutant Frequency Experiment 1b
Negative control	2.97	2.97
Positive control (Ethyl methansulfonate, 1 µL/mL)	942.47**	962.5**
400	2.02	2.02
550	5.15	4.12
700	4.12	4.12
800	5.1	3.06
900	4.17	4.17
1000	4.17	2.08
1100	3.13	3.13

 

Table 2: Mutant frequencies with S9-mix (5 hour treatment)

Concentration [µg/mL]	Mutant Frequency Experiment 1a	Mutant Frequency Experiment 1b
Negative control	2.97	3.96
Positive control (Dimethyl benz[a]nthracene, 20 µL/mL)	648.10**	629.11**
6.5	2.06	2.06
12.5	2.02	1.02
25	2.04	1.02
50	2.04	2.04
100	1.02	2.04
200	2.06	2.06
400	3.16	2.11

 

Table 3: Mutant frequencies without S9-mix (20 hour treatment)

Concentration [µg/mL]	Mutant Frequency Experiment 2a	Mutant Frequency Experiment 2b
Negative control	3.03	3.03
Positive control (Ethyl methansulfonate, 1 µL/mL)	987.32**	834.72**
300	2.04	1.01
350	2.04	3.06
400	3.09	3.09
450	2.06	4.17
500	3.13	2.08
550	2.11	3.16

 

Table 4: Mutant frequencies with S9-mix (5 hour treatment)

Concentration [µg/mL]	Mutant Frequency Experiment 2a	Mutant Frequency Experiment 2b
Negative control	2.97	2.97
Positive control (Dimethyl benz[a]nthracene, 20 µL/mL)	923.08**	916.67**
6.5	1.00	0.00
12.5	2.04	1.02
25	1.04	2.06
50	2.11	1.04
100	3.16	1.05
200	1.06	2.13
400	2.11	2.13**

**p < 0.01

Conclusions:

The test substance was not mutagenic in the HPRT test in CHO-K1 cells.

Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations in a preliminary study (without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1: 5-hour treatment period without S9 mix:

400, 550, 700, 800, 900,1000 and 1100 μg/mL

Experiment 1: 5-hour treatment period with S9 mix:

6.5, 12.5, 25, 50,100, 200 and 400 μg/mL

Experiment 2: 20-hour treatment period without S9 mix:

300, 350, 400, 450, 500, 550 and 600 μg/mL

Experiment 2: 5-hour treatment period with S9 mix:

6.5, 12.5, 25, 50,100, 200 and 400 μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation, when tested up to cytotoxic concentrations. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours) up to cytotoxic concentrations. Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency even when cytotoxicity occurred. Again, no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL).

Results of analytical measurement:

The test item concentrations of the test stock solutions were measured by reverse phase HPLC method with UV detection at the start of Main Mutation Assay, Experiment 1 and Experiment 2. The measured test item concentrations in the samples showed acceptable values of 93 % and 93 % in comparison to the nominal value.

The test substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.

Thus, the test item was not mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames-Test:

The test item Sika Hardener MI was tested for genetic toxicity in a bacterial reverse mutation assay according to EU Method B13/14 and 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 MI 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 (were mostly within the corresponding historical control data ranges, see: Validity of the Study). 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.

Significant, revertant colony number increases were observed in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA98, with addition of metabolic activation (+S9 Mix). The significant increases accompanied with dose-relationship, however did not reach the biological relevant threshold for being positive. No further biologically relevant increases (sporadic changes only) were observed in revertant colony numbers of any of the tester strains following treatment with Sika Hardener MI at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

In the Confirmatory Mutation Test following the pre-incubation procedure inhibitory effect of the test item was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains, without metabolic activation and included the lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were below the corresponding historical control data ranges) and slightly reduced background lawn development. No revertant growth was observed in S. typhimurium TA98 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 MI was considered non-mutagenic in this bacterial reverse mutation assay (ToxiCoop, 2011).

 

HPRT Test:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations in a preliminary study (without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

400, 550, 700, 800, 900,1000 and 1100 μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

6.5, 12.5, 25, 50,100, 200 and 400 μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

300, 350, 400, 450, 500, 550 and 600 μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

6.5, 12.5, 25, 50,100, 200 and 400 μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation, when tested up to cytotoxic concentrations. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours) up to cytotoxic concentrations. Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency even when cytotoxicity occurred. Again, no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL).

Results of analytical measurement:

The test item concentrations of the test stock solutions were measured by reverse phase HPLC method with UV detection at the start of Main Mutation Assay, Experiment 1 and Experiment 2. The measured test item concentrations in the samples showed acceptable values of 93 % and 93 % in comparison to the nominal value.

The test substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.

Thus, the test item was not mutagenic under the conditions of this study.

 

Chromosome Aberration Test:

The test item was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 400 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 50, 100, 200 and 250 μg/mL

with S9 mix: 25, 50, 100, 200 and 400 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 12.5, 25, 50, 100 and 150 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 12.5, 25, 50, 100 and 150 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 25, 50, 100, 200 and 400 μg/mL

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown as the concurrent positive controls, Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations. Based on these results, the substance was determined to not cause chromosomal aberrations.

Justification for classification or non-classification

Based on results of the in vitro genetic toxicity studies, SIKA Hardener MI was not classified and labelled as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No. 2017/776.