Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethyl]-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sika Hardener LJ was tested in three different in vitro genetic toxicity assays, with and without metabolic activation. The test item neither induces structural chromosome aberrations nor an increase in the mutant frequency in Chinese hamster ovary cells. The test item also did not induce gene mutations by base pair changes or frameshifts in the genome of bacteria.

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:

2004-11-30 - 2005-01-14

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:

July 21st, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 8th, 2000

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:

The Salmonella typhimurium histidine (his) reversion system measures his- -> 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- -> 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:

S9-mix (induction with Phenobarbitone and ß-naphthoflavone)

Test concentrations with justification for top dose:

Expermiment I (plate incorporation test) and II (pre-incubation test): 5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate.
Experiment III (confirmatory pre-incubation test): 50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Sika Hardener LJ formed a stable solution or a stable colloid system (an emulsion) in acetone.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 Migrated to IUCLID6: 9AA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without S9 Migrated to IUCLID6: NaN3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvr A without S9 Migrated to IUCLID6: MMS

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

TA 98, 100, 1535, 1537; E. coli WP2 uvr A with S9

Details on test system and experimental conditions:

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

DURATION
1. plate incorporation
The frozen cultures were thawed at room temperature and a measured amount was used for inoculating the over-night cultures in the assay. 200 µL inoculum was used for each 50 mL of broth. The bacterial strains were grown up in nutrient broth. The cultures were incubated for 10-14 h at 37 °C up to the late exponential or early stationary growth phase (approx. 109 cells/ml) in, a Gyrotory Water Bath Shaker. After the test item was added and mixed and after the solidification, the plates were inverted and incubated at 37°C for at least 48h in the dark. Revertant colonies were counted by hand.

2. preincubation

Histidine- Biotin overlay agar (for Salmonella typhimurium strains) contains per litre:

Agar Bacteriological 3.6 g
NaCl 4.5 g
D-Biotin (F.W. 244.3) 12.4 mg
L-Histidine- HCl H20 (F.W. 209,63) 10.5 mg

The agar solution was sterilised at 121 °C in an autoclave, and the Histidine-Biotin solution (0.5 mM) was sterilised by filtration through a 0.22 µm membrane filter

Tryptophan overlay agar (for Escherichia coli strain) contains per litre:

Agar Bacteriological 3.79 g
NaCl 4.74 g
Nutrient Broth 50.0 ml
L-Tryptophan (F.W. 204.23) 5.0 mg

The agar solution and the nutrient broth was sterilised at 121 °C in an autoclave. The Tryptophan solution (2 mg/mL) was sterilised by filtration through a 0.22 µm membrane filter.

NUMBER OF REPLICATIONS: The concentrations, including the controls, were tested in triplicates.

DETERMINATION OF CYTOTOXICITY

The toxicity of the test item was determined with strains TA 98 and TA 100 in a pre-experiment. 8 concentrations (5000.00; 2500.00; 1000.00; 316.205 100.00, 31.62, 10.00 and 3.162 µg/plate) were tested for toxicity and mutation induction with each 3 plates in the presence and absence of metabolic activation system (S9). The experimental conditions in this pre-experiment were the same as described for the main experiment I (plate incorporation test). Cytotoxic effects of the test item were observed at both examined test strains.

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 was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values were used for this calculation).

Evalution of Results

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
- the positive controls show a distinct enhancement over the control plate

A test item is considered 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 reversions is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
- if in strains TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvr A the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

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:

In order to investigate the potential of Sika Hardener LJ for its ability to induce gene mutations a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA.
Inhibitory, toxic effects of the test item were observed in all phases of the study. In experiment I the inhibitory effect of the test item was observed in case of Salmonella typhimurium TA 100, TA 1535 and TA 1537. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in the concentration range of 5000.00-158.11 µg/plate in S. typhimurium TA 1537 and in the concentration range of 5000.00-500.00 µg/plate in TA 100 and TA 1535. In absence of metabolic activation the revertant colony numbers were reduced only in case of TA 1537 in the concentration range of 5000.00-500.00 µg/plate. In S. typhimurium TA 100 the appearance of pinpoint colonies (concentration range: 5000.00-1581.14 µg/plate (+S9)) and in TA 1537 reduced background lawn development (5000.00 µg/plate (±S9), 5000.00-1581.14 µg/plate (+S9)) was also observed. In the experiment II using the pre-incubation method, the inhibitory effect manifested much stronger. The pre-incubation method is more sensitive than the plate incorporation assay. The revertant colony numbers compared to the solvent control plates as well as the background lawn development were reduced in the investigated Salmonella typhimurium strains. The appeared small pinpoint colonies (inall Salmonella typhimurium strains) were also signs of inhibition. The inhibition manifested stronger in the activation part (+S9) of the experiment at higher concentration levels (5000.00-158.11 µg/plate, at TA 1535 5000.00-500.00 µg/plate) with respect to the reduction of revertant colony numbers, but inhibitory effects could be observed down to lower concentration levels in the non-activation part of the experiment in all cases. In the test strains Salmonella typhimurium TA 100, TA 1535 and TA 1537 the toxicity affected even the lowest concentration (5.00 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA.
In the additional confirmatory mutation (experiment III) assay using the preincubation method the results of the experiment II were confirmed. The examined strains Salmonella typhimurium TA 100, TA 1535 and TA 1537 were inhibited without metabolic activation at the concentration range of 50.00-5.00 µg/plate. At the lower concentrations no cytotoxic effects were noted.


TEST-SPECIFIC CONFOUNDING FACTORS: none

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects of the test item were observed at both examined test strains. The revertant colony numbers were slightly reduced compared to the solvent control plates in case of Salmonella typhimurium TA 98 in the concentration range of 5000.00-2500.00 µg/plate, at TA 100 in the concentration range of 5000.00-316.20 µg/plate in presence of metabolic activation system. Beside the reduced revertant colony numbers compared to the solvent control plates reduced background lawn development and appearance of pinpoint colonies was observed as sign of the toxic effect of the test item in Salmonella typhimurium TA 100. The test item did not show mutagenic effect on the examined bacterium strains. The number of the revertant colonies was not significantly enhanced. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon in the concentration range of 5000.00-1000.00 µg/plate.

Conclusions:

Based on the results of this Ames test, Sika Hardener LJ is considered to be non-mutagenic with or without metabolic activation in bacterial systems.

Executive summary:

Sika Hardener LJ was assessed in an Ames test according to EU method B.13/14 OECD guideline 471. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvr A were testet in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at seven concentrations each (5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate). An additional confirmatory test (experiment III) was carried out, because of the noted toxicity in the experiment II. The examined bacterium strains were; Salmonella typhimurium TA 100, TA 1537 and TA 1535. The experiment was performed in the absence of a post-mitochondrial supernatant (S9). In this confirmatory experiment the preincubation method was also used five different concentrations (50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate).

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with Sika Hardener LJ 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 solvent control values were observed in all experimental phases of the study. However, there was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in the performed experiment. The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate. The reported data of this mutagenicity assay show that, under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Sika Hardener LJ is considered to be 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:

2005-02-22 - 2005-05-15

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:

July 21st 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 8th 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham`s F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, before freezing at -80 °C

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix (phenobarbitone and ß naphtoflavone induced)

Test concentrations with justification for top dose:

Experiment A
without S9 mix: 4 h treatment, 20 h preparation interval
10, 40, 75, 100, 200 µg/mL Sika Hardener LJ
with S9 mix: 4 h treatment, 20 h preparation interval
10, 40, 75, 100, 200 µg/mL Sika Hardener LJ

Experiment B
without S9 mix: 20 h treatment, 28 h preparation interval
6, 12, 20, 30, 40 µg/mL Sika Hardener LJ
with S9 mix: 4 h treatment, 28 h preparation interval
10, 40, 75, 100, 200 µg/mL Sika Hardener LJ

Vehicle / solvent:

Acetone, because it is compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: EMS

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: Experiment A: with and without S9-Mix, 4h; Experiment B: without S9 Mix, 20h; with S9 Mix, 4 h

SPINDLE INHIBITOR: Colchicine (0.2 µg/mL
STAIN: 10% Giemsa at pH 6.8

NUMBER OF REPLICATIONS: all treatment groups and controls were run in triplicate.

NUMBER OF CELLS EVALUATED: 200 metaphase cells containing 2N±2 cetromeres from each experimental group

DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency (% relative survival)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The criteria for determining a positive result are:
- a clear and concentration-related increase in the number of cells with aberrations
- biologically relevant response for at least one of the dose groups, higher (two-three times) than the laboratory negative control range (up to 5 % aberrant cells)
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both biological and statistical significance should be considered together. A test item for which the results do not meet the above criteria is considered non-mutagenic in this system.

Statistics:

NA

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

Solubility and Dose Selection

Sika Hardener LJ was dissolved for treatment in Acetone. There was no precipitation in the medium at all concentrations tested. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for Chromosome Aberration Assays, both in the absence and in the presence of S9 mix metabolic activation system. Toxicity was determined by assessment of the relative plating efficiency (% relative survival). Four-hour treatment with Sika Hardener LJ in the absence of S9 mix resulted in 97.20 % relative survival at 5 µg/mL, 44.57 % at 100 µg/mL and 2.14 % relative survival at 500 µg/mL concentrations. When the four-hour treatment was performed in the presence of metabolic activation the relative survival percentages were 97.64 % at the 5 µg/mL concentration and 41.92 % at 100 µg/mL concentration. At 500 µg/mL none of the cells survived. Twenty-hour treatment of Sika Hardener LJ in the absence of S9 mix caused 97.70 % relative survival at 5 µg/mL concentration, 67.76 % at 25 µg/mL and 0.49 % relative survival at 50 µg/mL concentration. These results were used to select the concentrations of Sika Hardener LJ for Chromosome Aberration Assays.

Chromosome Aberration Assay

The cytotoxicity at the highest concentrations was adequate in the studies as indicated by the reduction of % cell survival of at least 50 %. In the (untreated) control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 % proving the suitability of the cell line used. Ethylmethane sulphonate (0.4 µL/mL) and N-Nitrosodimethylamine (0.4 µL/mL) as positive controls functioned well and resulted in the expected biologically relevant increase of cells with structural chromosome aberrations.

The studies are, therefore, considered as valid and reliable.

In Experiment A , the Sika Hardener LJ did not induce an increase in the number of cells with aberrations without gaps at any examined concentrations, either in the absence or in the presence of metabolic activation up to and including cytotoxic concentrations. In this experiment the number of aberrant cells after treatment with 75 and 200 ug/ml of test item in the presence of metabolic activation were slightly above the upper range of the historical control data. This slight and non-dose-related higher frequency of the cells with structural chromosome aberrations was not biologically important.

In Experiment B, a four-hour treatment with Sika Hardener LJ in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps at 10, 40, 75, 100 and 200 µg/mL further indicating that the findings in Experiment A were within the normal biological variation. When the Sika Hardener LJ was examined (6, 12, 20, 30 and 40 µg/mL) without S9 mix, with a long treatment period (20 hours) the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control.

No biologically relevant increase in the rate of polyploid and endoreduplicat metaphases was found after treatment with the different concentrations of Sika Hardener LJ.

Conclusions:

Sika Hardener LJ was tested both with and without metabolic activation and did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells. Therefore, Sika Hardener LJ is considered negative in this system.

Executive summary:

Sika Hardener LJ was tested in a Chromosome Aberration Assay in CHO-KI cells according to the OECD guideline 273. In two independent assays both in the absence and presence of metabolic activation no biologically significant increase in the number of cells with structural chromosome aberrations without gaps was noted in the presence of the test compound. There was no biologically relevant increase in the rate of polyploid and endoreduplicated metaphases.The validity of the test was shown by the use of Ethylmethane sulphonate (0.4 µL/mL) and N-Nitrosodimethylamine (0.4 µL/mL) as positive controls. Therefore Sika Hardener LJ is considered negative in this system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-09 to 2013-05-09

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

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1

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 %).
- Properly maintained: yes
- Checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

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 1, 5-hour treatment period without S9 mix: 150, 200, 250, 275, 300 and 325 μg/mL
Experiment 1, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 μg/mL
Experiment 2, 20-hour treatment period without S9 mix: 100, 150, 200, 250, 275, 300 and 325 μg/mL
Experiment 2, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: The vehicle was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

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:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (+/- S9 mix) and 20 hours (-S9 mix)
- Expression time: 8 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: one

NUMBER OF CELLS EVALUATED: 1E+06 cells: (5 plates at 2 x 1E+05 cells/plate)

DETERMINATION OF CYTOTOXICITY
- Method: colony forming efficiency, relative total growth

Evaluation criteria:

The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (1E+06 cells: 5 plates at 2 x 1E+05 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 1E+06 clonable cells.

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)

Remarks:

K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: yes
- Precipitation: not observed up to the highest test item concentration tested

RANGE-FINDING/SCREENING STUDIES:
Sika Hardener LJ was dissolved in DMSO. A clear solution was obtained up to a concentration of 25 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.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the positive and negative control cultures were consistent with the historical control data.

Results of the concentration selection cytotoxicity assay 

Test group	Dose [µg/mL]	S9 mix	Treatment time [h]	Relative survival [% control]
Untreated control	-	-	5	100
Solvent control	-	-	5	100
SIKA Hardener LJ	10	-	5	100
SIKA Hardener LJ	25	-	5	100
SIKA Hardener LJ	50	-	5	99
SIKA Hardener LJ	100	-	5	100
SIKA Hardener LJ	150	-	5	96
SIKA Hardener LJ	200	-	5	90
SIKA Hardener LJ	250	-	5	70
SIKA Hardener LJ	300	-	5	31
Untreated control	-	+	5	118
Solvent control	-	+	5	100
SIKA Hardener LJ	10	+	5	100
SIKA Hardener LJ	25	+	5	92
SIKA Hardener LJ	50	+	5	96
SIKA Hardener LJ	100	+	5	70
SIKA Hardener LJ	150	+	5	47
SIKA Hardener LJ	200	+	5	38
SIKA Hardener LJ	250	+	5	30
SIKA Hardener LJ	300	+	5	7
Untreated control	-	-	20	100
Solvent control	-	-	20	100
SIKA Hardener LJ	5	-	20	99
SIKA Hardener LJ	10	-	20	100
SIKA Hardener LJ	25	-	20	100
SIKA Hardener LJ	50	-	20	99
SIKA Hardener LJ	100	-	20	98
SIKA Hardener LJ	150	-	20	85
SIKA Hardener LJ	200	-	20	79
SIKA Hardener LJ	250	-	20	56

 

Conclusions:

Sika Hardener LJ 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. Thus, Sika Hardener LJ was not mutagenic under the conditions of this study.

Executive summary:

The test item, SIKA Hardener LJ 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).

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: 150, 200, 250, 275, 300 and 325 μg/mL

Experiment 1, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 μg/mL

Experiment 2, 20-hour treatment period without S9 mix: 100, 150, 200, 250, 275, 300 and 325 μg/mL

Experiment 2, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 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. 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). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

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.

SIKA Hardener LJ 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. Thus, SIKA Hardener LJ 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

Chromosome aberration test

Sika Hardener LJ was tested in a Chromosome Aberration Assay in CHO-KI cells according to the OECD guideline 273. In two independent assays both in the absence and presence of metabolic activation no biologically significant increase in the number of cells with structural chromosome aberrations without gaps was noted in the presence of the test compound. There was no biologically relevant increase in the rate of polyploid and endoreduplicated metaphases.The validity of the test was shown by the use of Ethylmethane sulphonate (0.4 µL/mL) and N-Nitrosodimethylamine (0.4 µL/mL) as positive controls. Therefore Sika Hardener LJ is considered negative in this system.

Ames Test

Sika Hardener LJ was assessed in an Ames test according to EU method B.13/14 OECD guideline 471. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvr A were testet in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at seven concentrations each (5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate). An additional confirmatory test (experiment III) was carried out, because of the noted toxicity in the experiment II. The examined bacterium strains were; Salmonella typhimurium TA 100, TA 1537 and TA 1535. The experiment was performed in the absence of a post-mitochondrial supernatant (S9). In this confirmatory experiment the preincubation method was also usedfive different concentrations (50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate).

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with Sika Hardener LJ 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 solvent control values were observed in all experimental phases of the study. However, there was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in the performed experiment. The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate. The reported data of this mutagenicity assay show that, under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Sika Hardener LJ is considered to be non-mutagenic in this bacterial reverse mutation assay.

HPRT Test

The test item, SIKA Hardener LJ 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).

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: 150, 200, 250, 275, 300 and 325 μg/mL

Experiment 1, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 μg/mL

Experiment 2, 20-hour treatment period without S9 mix: 100, 150, 200, 250, 275, 300 and 325 μg/mL

Experiment 2, 5-hour treatment period with S9 mix: 50, 100, 150, 200, 250, 275, and 300 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. 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). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

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.

SIKA Hardener LJ 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. Thus, SIKA Hardener LJ was not mutagenic under the conditions of this study.

 

In conclusion, SIKA Hardener LJ is considered non-mutagenic in vitro.

Justification for classification or non-classification

Based on the results of three different in vitro genotoxicity studies, Sika Hardener LJ was not classified and labelled as genotoxic according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.