Reaction product of propylidynetrimethanol, propoxylated, reaction products with ammonia and 2,2-Dimethyl-3-(4-morpholinyl)propanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Sika Hardener MTJ did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in the available bacterial reverse mutation assay (Ames test). The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in an 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. (HPRT) The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, the test item is considered as not clastogenic in this system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-29 to 2014-07-23

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-naphtoflavone induced rat liver S9 Mix

Test concentrations with justification for top dose:

Experiment I, without S9-Mix:
1000, 1200, 1400, 1600, 1800, 2000, 2200 µg/mL
With S9-Mix:
100, 150, 200, 300, 400, 500, 600 µg/ mL

Experiment 2, without S9-Mix:
1000, 1100, 1200, 1300, 1350, 1400, 1500 µg/mL
Experiment 2, With S9 Mix:
100, 150, 200, 300, 400, 500, 600 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Both experiments without S9-Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Both experiments with S9-Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours, 20 hours
- Expression time (cells in growth medium): 8 days
- Fixation time: After selection period.

SELECTION AGENT: EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL containing 3.4 μg/mL of 6-thioguanine (6-TG)).

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 200

STAIN: Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: Determining the relative cloning efficiency (survival).

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:

See "Any other information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and Dose selection

The test item was dissolved in DMSO. A clear solution was obtained up to a concentration of 150 mg/mL. For all test item concentrations examined, no precipitation in the medium was noted. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays (Experiments 1 and 2), both in the absence and in the presence of a metabolic activation system (rodent liver S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The results obtained were used for dose selection of the test item in the Main Mutation Assays.

Main Experiment I

On Day 1, there was very clear evidence of toxicity with the test item in both absence and presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
In Experiment 1, in some cases the mutant frequencies of the cells were slightly above the concurrent control values. These higher values did not show biologically or statistically significant alterations compared to the concurrent control.

Main Experiment II

There was very clear evidence of toxicity with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls and very clear evidence of toxicity in the absence of metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. On the basis of Day 8 cloning efficiency data the cells had recovered during the expression period.
In Experiment 2, in some cases the mutant frequencies of the cells were slightly above the concurrent control values. These higher values 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). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (0.4 or 1.0 ìL/mL) and 7,12-Dimethyl benz[a]anthracene (20 ìg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.
The osmolality of test item solutions did not show any alterations compared to the concurrent control groups in Experiments 1 and Experiment 2. The pH values of test item solutions with S9 mix were similar compared to the control values in Experiments 1 and Experiment 2. The pH values of test item solutions without S9 mix were slightly above the concurrent control values in Experiments 1 and Experiment 2.

Conclusions:

negative

The test item 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.

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 made 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 as follows:

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

1000, 1200, 1400, 1600, 1800, 2000 and 2200 μg/mL

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

100, 150, 200, 300, 400, 500 and 6001 μg/mL

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

1000, 1100, 1200, 1300, 1350, 1400 and 1500 μg/mL

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

100, 150, 200, 300, 400, 500 and 6001 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.

As in Experiment 1, in Experiment 2 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 and 7,12-Dimethyl benz[a]anthracene.

The test item tested both without and with metabolic activation, did not induce increases in mutant frequency 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-29 to 2014-07-23

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:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S9 Mix

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 900, 1200, 1500, 1800 and 2000 μg/mL
with S9 mix: 300, 800, 1300 1800 and 2400 μg/mL
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 100, 300, 600, 900 and 1100 μg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 100, 300, 600, 900 and 1100 μg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 300, 800, 1300 1800 and 2400 μg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide monohydrate

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 3 hours and 20 hours
- Fixation time: 20 hours and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Cell cultures were treated with colchicine (0.2 μg/mL) 2.5-3 hours prior to harvest.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 400 metaphases

STAIN: Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.

OTHER EXAMINATIONS:
- Other: The changes in pH and osmolality of the test media were determined for every treatment in Experiment A and Experiment B.
- Additionally the numbers of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN (1985), and Savage (1976, 1983).

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and dose selection

The test item was dissolved in DMSO. A clear solution was obtained up to a concentration of 150 mg/mL. There was no precipitation in the medium at any concentration tested. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting, based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. These results were used to select concentrations of the test item for the Chromosome Aberration Assays. Based on the results of the cytotoxicity assay, the following concentrations were selected ranging from little to maximum (< 50 % survival) toxicity and evaluated in the main studies (Experiment A and B). All concentrations were run in duplicates (incl. negative and positive controls) and at least 400 wellspread metaphases were assessed.

Chromosome aberration test

The cytotoxicity at the highest concentrations was adequate in the studies. In Experiment A, the test item did not induce an increase in the number of cells with aberrations at any examined concentration, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistically significant differences between treatment and control groups and no dose-response relationship was noted. In Experiment B, the test item was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations did not show significant alterations compared to the concurrent solvent controls. There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).
A 3-hour treatment 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 over concurrent solvent control.
As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data.
Polyploid and endoreduplicated metaphases were not found after treatment with the different concentrations of the test item.
In Experiments A and B, the pH and osmolality of negative control and treatment solutions were measured. There were no significant differences between treatment and negative control groups.
In the concurrent solvent control group the percentage of cells with structural aberration(s) without gap was equal to or less than 5 %, proving the suitability of the cell line used.
The concurrent positive controls Ethyl methanesulfonate (0.4 and 1.0 µL/mL) and Cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls. Thus, the study is considered as valid.

Conclusions:

negative

The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, the test item is considered as not clastogenic in this system.

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 made 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: 900, 1200, 1500, 1800 and 20001 μg/mL

with S9 mix: 300, 800, 1300 1800 and 2400 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 100, 300, 600, 900 and 11001 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 100, 300, 600, 900 and 11001 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 300, 800, 1300 1800 and 24001 μg/mL

These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations. 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.

The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-20 to 2013-04-2013

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:

21 July 1997

Deviations:

no

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

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:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Experiment I: 5000; 1581, 500, 158, 50 and 15.8 μg/plate
Experiment II: 5000; 1581, 500, 158, 50 and 15.8 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO, Ultrapure water
- Justification for choice of vehicle: The vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

Salmonella TA98 (-S9 mix)

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella TA100 and TA1535 (-S9 mix)

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Salmonella TA1537 (-S9 mix)

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2 uvrA (-S9 mix)

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Salmonella TA98, TA100, TA1535, TA 1538 and E.coli (+S9 mix)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min at 37 ºC
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT:
- Salmonella typhimurium: L-Histidine
- Escherichia coli: L-Tryptophan

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertants of vehicle control

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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay 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:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 5000 µg/tube revealed test item particles in the agar mix. A clear solution was achieved at all concentrations below 5000 µg/tube.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
Revertant colony numbers below the vehicle and corresponding historical control data ranges, furthermore affected background lawn development (slightly reduced background lawn development) showed the inhibitory effect of the test item in S. typhimurium TA100 at 5000 μg/plate, without metabolic activation (-S9 Mix).
The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control (and below the historical control data range) without any biological significance in S. typhimurium TA100 at 500 μg/plate (-S9 Mix).
The revertant colony numbers remained in the vehicle control data range, however were below the corresponding historical control data range (as reflecting the biological variability range of the applied test system) in S. typhimurium TA100 at 1581 μg/plate and at 5 μg/plate, without metabolic activation (-S9 Mix).
The revertant colony numbers were slightly lower (but remained in the corresponding historical control data range) than the revertant colony numbers of the vehicle control plates in the case of S. typhimurium TA98, at 5000 μg/plate, without metabolic activation (-S9 Mix), in TA100, at 5000 μg/plate and 5 μg/plate, with addition of metabolic activation (+S9 Mix).
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at the concentrations of 5000 and 1581 μg/plate, with addition of metabolic activation (+S9 Mix).

COMPARISON WITH HISTORICAL CONTROL DATA:
Valid tests were performed since the tester strains (used in this study) demonstrated the specific phenotype characteristics, agreed with the corresponding historical control data ranges, and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity (according to the provided Certificates) and was active in the applied system (2AA treatments).
Each of the investigated reference mutagens showed the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases. The revertant colony numbers of the reference mutagens were within the corresponding historical control data ranges.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates were in the actual historical control data ranges in all experimental phases.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Initial Mutation Test
As sign of the inhibition, the lower revertant colony numbers (compared to the revertant colony numbers of the vehicle control) were below the corresponding historical control data range in S. typhimurium TA100, at 1581 μg/plate (±S9 Mix), furthermore at 500 μg/plate (-S9 Mix).
Confirmatory Mutation Test
As sign of the inhibition, the revertant colony numbers (within the historical control data ranges) were significantly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA1537 at 1581 μg/plate (+S9 Mix).

Conclusions:

negative with metabolic activation
negative without metabolic activation

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 tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item 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.

The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the tested concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

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 and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item 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 mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

Inhibitory effect of the test item was observed in the examined Salmonella typhimurium strains. The inhibition appeared unequivocally down to and including the concentration of 500 μg/plate in Salmonella typhimurium TA100 in both tests, in TA1537 in the Confirmatory Mutation Test in absence, and down to 1581 μg/plate in TA100 in the Initial Mutation Test, in TA1537 in the Confirmatory Mutation Test, in presence of exogenous metabolic activation.

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 tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item Sika Hardener MTJ 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. The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the tested concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate. 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 and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item 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 mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Inhibitory effect of the test item was observed in the examined Salmonella typhimurium strains. The inhibition appeared unequivocally down to and including the concentration of 500 μg/plate in Salmonella typhimurium TA100 in both tests, in TA1537 in the Confirmatory Mutation Test in absence, and down to 1581 μg/plate in TA100 in the Initial Mutation Test, in TA1537 in the Confirmatory Mutation Test, in presence of exogenous metabolic activation. 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 tester strains used. Therefore, the test item is considered non-mutagenic in the bacterial reverse mutation assay available.

 

In Vitro Mammalian Cell Gene Mutation Test (HPRT Assay):

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 made 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 as follows:

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

1000, 1200, 1400, 1600, 1800, 2000 and 2200 μg/mL

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

100, 150, 200, 300, 400, 500 and 6001 μg/mL

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

1000, 1100, 1200, 1300, 1350, 1400 and 1500 μg/mL

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

100, 150, 200, 300, 400, 500 and 6001 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. As in Experiment 1, in Experiment 2 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).

The test item tested both without and with metabolic activation, did not induce increases in mutant frequency 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 Sika Hardener MTJ 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 made 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: 900, 1200, 1500, 1800 and 20001 μg/mL

with S9 mix: 300, 800, 1300 1800 and 2400 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 100, 300, 600, 900 and 11001 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 100, 300, 600, 900 and 11001 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 300, 800, 1300 1800 and 24001 μg/mL

 

These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations. 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.

The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.