Dodecanoic acid, 1,1'-[1,6-hexanediylbis[nitrilo(2,2-dimethyl-1-propanyl-3-ylidene)]] ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

SIKA Hardener LH was tested in three different in vitro genetic toxicity studies, both with and without metabolic activation. SIKA Hardener LH did not induce gene mutations by frameshift or base-pair substitution in an Ames test. SIKA Hardener LH did not induce structural chromosome aberrations in Chinese Hamster lung cells and was therefore not considered clastogenic in the tested system. Additionally, SIKA Hardener LH showed no mutagenic effect in an HPRT assay. Overall, SIKA Hardener LH was considered non genotoxic.

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:

2004-11-26 to 2005-01-28

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:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion 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:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S9-mix from rats induced with Phenobarbitone (PB) and ß-naphthoflavone (BNF)

Test concentrations with justification for top dose:

5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate.
In case of the tester strains Salmonella typhimurium TA100, TA 1535 and TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) in experiment II.
Therefore, an additional (third) confirmatory test was carried out with Salmonella typhimurium TA 100, TA 1535 and TA 1537 and the following concentrations were tested: 50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate; MMS

Remarks:

E. coli WP2 uvrA without S-9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

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

Remarks:

S. typhimurium: TA 98 without S-9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium: TA 100; TA 1535 without S-9 Migrated to IUCLID6: NaN3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

S. typhimurium TA 1537 without S-9 Migrated to IUCLID6: 9-AA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 2-AA

Remarks:

S. typhimurium: TA 100; TA 98; TA 1535; TA 1537 and E. coli WP2 uvrA with S-9

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number

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).

Evaluation 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 uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

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).

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate.

RANGE-FINDING/SCREENING STUDIES: Inhibitory, toxic effects of the test item were observed in all phases of the study. In experiment I the inhibitory effect was observed in all Salmonella typhimurium strains. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in S. typhimurium TA 98 and TA 1535 at the highest concentration level of 5000.00 g/plate, in S. typhimurium TA 100 in the concentration range of 5000.00-500.00 µg/plate, and in S. typhimurium TA 1537 in the concentration range of 5000.00-1581.14 µg/plate. The revertant colony numbers were also reduced without metabolic activation: In Salmonella typhimurium TA 100 in the concentrations of 5000.00 and 1581.14 µg/plate and in TA 1537 in the concentration range of 5000.00-158.11 µg/plate. Background lawn reduction was detected in TA 100 (+S9; at 5000.00 and 1581.14 µg/plate), in TA 1535 (+S9; at 5000.00 µg/plate) and in TA 1537 (+/-S9; at 5000.00, 1581.14 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA. 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 and small pinpoint colonies appeared in the investigated Salmonella typhimurium strains.

The inhibition manifested stronger in the activation part (+S9) of the experiment at higher concentration levels (5000.00-158.11 µg/plate) with respect to the reduction of revertant colony numbers, but the inhibitory effect 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 TA100, TA 1535 and TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) while in case of TA 98 it was observable down to the concentration preceding the lowest concentration (15.81 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA.

In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. The examined strains were inhibited without metabolic activation at the concentration range of 50.00-5.00 µg/plate. The inhibition manifested in reduction of revertant colony numbers, in reduction of background lawn development and in appearance of pinpoint colonies. At the lower concentrations no cytotoxic effects were noted.

HISTORICAL CONTROL DATA:
- Historical control values for spontaneous revertants per plate are as follows: (-S9) Salmonella typhimurium TA 98: 15-60, TA 100: 75-200, TA 1537: 3-30, TA 1535: 3-28 and Escherichia coli WP2 uvrA: 8-50.

Conclusions:

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

Executive summary:

SIKA Hardener LH was assessed in an Ames test according to EU method B.13/14, OECD guideline 471 and EPA OPPTS 870.5100. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were tested in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). Two independent experiments were run (Initial Mutation Assay and Confirmatory Mutation Assay) up to limit or cytotoxic concentrations, with and without metabolic activation (S9 mix). All assays, including controls, were tested in triplicate.

Inhibitory effects of treatment were observed in all phases of the study. In the experiment I the inhibitory effect appeared in reduction of spontaneous rate compared to the solvent control values (Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537), and in background lawn reduction (Salmonella typhimurium TA 100, TA 1535 and TA 1537). In the experiment II revertant colony numbers compared to the solvent control plates as well as the background lawn development were reduced and small pinpoint colonies appeared in the investigated Salmonella typhimurium strains. Reduction of revertant colony numbers was pronounced at high concentration levels (5000.00-158.11 µg/plate) with metabolic activation, but observed down to lower concentrations in non-activated treatments. In test strains Salmonella typhimurium TA100, TA 1535 and TA1537 cytotoxic effects were observed even at lowest concentrations (5.00 µg/plate) and for TA 98 observed down to 15.81 µg/plate. No cytotoxic effects were observed in Escherichia coli WP2uvrA. In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. Cytotoxic effects were observed at concentrations of 50.00-5.00 µg/plate without metabolic activation. At the lower concentrations no cytotoxic effects were noted.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with SIKA Hardener LH 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.

The 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 LH 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:

2005-02-21 to 2005-07-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

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

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):

CELLS USED
- Source of cells: American Type Cell Culture Collection (USA)
- doubling time: 12 -14 hours
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Methods for maintenance in cell culture: Cell cultures are maintained in exponential growth by sub-culturing twice a week at 37° in a humified atmosphere containing 5% CO2
- Modal number of chromosomes: 20-22

MEDIA USED
- Type and identity of media including CO2 concentration: Ham’s F12 medium supplemented with L-gluatamine (1 mM), penicillin (50-100 units/mL), streptomycin (50 µg/mL) and inactivated foetal calf serum (final conc. 10%) ; serum content was reduced to 0% and 5% during 4 hours treatment and 20 hours treatment, respectively
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver induced by Phenobarbitone and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment A
without S9 mix: 4-h treatment, 20-h preparation interval
50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL Sika Härter LH (VP)
with S9 mix: 4-h treatment, 20-h preparation interval
50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL Sika Härter LH (VP)

Experiment B
without S9 mix: 20-h treatment, 28-h preparation interval
20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL and 150 µg/mL Sika Härter LH (VP)
with S9 mix: 4-h treatment, 28-h preparation interval
50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL Sika Härter LH (VP)

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

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
- Cell density at seeding: 2 x 10E+6 cells

DURATION
- Exposure duration: 4 hours and 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours (Experiment A), 28 hours (Experiment B)

SPINDLE INHIBITOR: (Colchicine (0.2 µL/mL)

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were swollen with 0.075 M KCL solution, then washed three times in fixative, (3:1 mixture of methanol: acetic-acid until preparation becomes plasma free) and dropped onto slides and air-dried.

NUMBER OF CELLS EVALUATED: 200 cells per dose

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

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

Evaluation criteria:

Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) were recorded.
– Individual culture data were summarised in tabular form.
– Equivocal results were clarified by further testing preferably using modification of experimental conditions.

Interpretation of Results
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 (at least two- three times) than the laboratory negative control range.
According 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

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: Toxicity was determined by assessment of the relative plating efficiency (% relative survival). Four-hour treatment with Sika HardenerLH (VP) in the absence of S9 mix resulted in 100.00 % relative survival at 10 µg/mL and 9.06 % relative survival at 1000 µg/mL concentration. When the four-hour treatment was performed in the presence of metabolic activation the relative survival percentages were 99.51 % at the 10 µg/mL concentration and 8.87 % at 1000 µg/mL concentration. Twenty-hour treatment of Sika Härter LH (VP) in the absence of S9 mix caused 99.51 % relative survival at 10 µg/mL concentration and 21.19 % relative survival at 100 µg/mL concentration

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Conclusions:

Sika Hardener LH (VP) tested both with and without metabolic activation did not induce structural chromosome aberrations in the test in Chinese Hamster ovary cells. Therefore, Sika Härter LH (VP) is considered negative in this system.

Executive summary:

Test item SIKA Hardener LH was tested in a Chromosome Aberration Assay in CHO-KI cells according to EU method B.10, OECD guideline 473 and EPA OPPTS 870.5375. The test item was dissolved in Acetone and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A

without S9 mix: 4-h treatment, 20-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

with S9 mix: 4-h treatment, 20-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

Experiment B

without S9 mix: 20-h treatment, 28-h preparation interval

20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL and 150 µg/mL SIKA Hardener LH

with S9 mix: 4-h treatment, 28-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

The highest concentrations of test item SIKA Hardener LH (80, 150 µg/mL in the absence of S9 mix and 600 µg/mL in the presence of S9 mix) were cytotoxic and no viable cells remained. In Experiment A no significant increases in the number of cells showing structural chromosome aberrations were observed up to and including cytotoxic concentrations in the absence of metabolic activation. In the presence of metabolic activation the test item (200 µg/mL and 400 µg/mL) slightly increased the frequency of the cells with structural chromosome aberrations when compared to the concurrent control and when compared to historical control. This slightly higher frequency of the cells with structural chromosome aberrations was not biologically significant as it was noted at cytotoxic concentrations and was not observed in Experiment B.

In Experiment B no increase in the frequency of cells with structural chromosome aberrations (50 µg/mL, 100 µg/mL, 200 µg/mL and 400 µg/mL) was observed. When assessed without metabolic activation over a treatment period of 28 hours the frequency of the cells with structural chromosome aberrations was comparable to the concurrent control up to and including cytotoxic concentrations.

In Experiment B there were no significant increases in the number of cells showing structural chromosome aberrations without gaps, up to and including cytotoxic concentrations either in the absence or in the presence of metabolic activation.

There was no biologically relevant increase in the rate of polyploid and endoreduplicated metabphases in either experiment in the presence or in the absence of metabolic activation. 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.

In summary, SIKA Hardener LH tested both with and without metabolic activation did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells. Therefore, SIKA Hardener LH was considered not clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-01-2011 to16-02-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Version / remarks:

1997

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:

The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures)
- Suitability of cells: V79 cells are well established and stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).
- Doubling time: 12 -14 h
- Methods for maintenance in cell culture: The laboratory cultures were maintained in 75 cm² plastic flasks at 37 °C in an incubator with a humidified atmosphere, set at 5 % CO2.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium (DME-10) supplemented with 1 % of antibioticantimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 and 20 hours treatments with test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (DME-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

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 and with S9 mix:
39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

Experiment 2,
20-hour treatment period without S9 mix:
19.6, 39.1, 78.2 104.2, 130.2, 156.3 and 234.5 μg /mL

Experiment 2,
5-hour treatment period with S9 mix:
39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

Vehicle / solvent:

N,N-Dimethylformamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 10E+6 cells per dish

DURATION
- Exposure duration: 5 hours (Experiment 1); 5 hours and 20 hours (Expriment 2)
- Expression time (cells in growth medium): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

NUMBER OF CELLS EVALUATED: 2x10E+5 cells/dish

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: 200 cells were used

Evaluation criteria:

Calculation of mutation frequency:
- The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (106 cells: 5 plates at 2x105 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 106 clonable cells.

Assay acceptance criteria:
- The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.
- The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency.
- The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 0 and 70% to 130% on Day 7.

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 is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is 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 is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: ph values of untreated and test item treated cells were in a comparable range
- Effects of osmolality: osmolality of untreated and test item treated cells were in a comparable range
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: 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. In Experiments 1 and 2 in the absence and in the presence and in Experiment 2 in the presence of metabolic activation the upper test item dose level selected was 937.5 μg Sika Härter LH /mL. In Experiment 2 in the absence of metabolic activation the upper test item dose level was 234.5 μg Sika Härter LH /mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: - S9: min = 328.78, max = 1075.00, mean = 619.93, SD = 217.85; + S9: min = 275.86, max = 508.00, mean = 327.65, SD = 61.98
- Negative (solvent/vehicle) historical control data: - S9: min = 3.88, max = 14.14, mean = 6.93, SD = 2.56; + S9: min = 2.97, max = 13.40, mean = 6.92, SD = 2.60

Conclusions:

No mutagenic effects of SIKA Hardener LH were observed in presence or absence of metabolic activation under conditions of testing.

Executive summary:

SIKA Hardener LH was tested in a Mammalian Gene Mutation Test in V79 cells. The test item was dissolved in N,N-Dimethylformamide and the following concentrations were selected on the basis of preliminary cytotoxicity investigations, without and with metabolic activation using S9 mix. Two independent experiments (both run in duplicate) with at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum toxicity (< 50 % survival):

Experiment 1

5-hour treatment period without and with S9 mix:

39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

Experiment 2

20-hour treatment period without S9 mix:

19.6, 39.1, 78.2 104.2, 130.2, 156.3 and 234.5 μg /mL

5-hour treatment period with S9 mix:

39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

In Experiment 1, there was a slightly higher mutation frequency at 39.1, 78.2, 312.5, 468.8, 625 and 937.5 μg/mL in the absence of metabolic activation and 39.1, 78.2, 156.3, 468.8, 625 and 937.5 μg/mL in the presence of metabolic activation compared to the concurrent control. These alterations were not biologically or statistically significant and no dose-response relationships were noted.

In Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted. The mutant frequency of the cells was slightly higher compared to the concurrent negative (solvent) control, but these alterations were not statistically significant when observed over a prolonged treatment period of 20 hours in the absence of metabolic activation. At all concentrations the mutant frequencies were above of negative control values when the test item was observed in the presence of metabolic activation, but these alterations were not toxicologically significant, further indicating that the findings in Experiment 1 were within the normal biological variation.

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 LH tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster lung cells. SIKA Hardener LH was not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reverse Mutation Assay using Bacteria (Ames test)

SIKA Hardener LH was assessed in an Ames test according to EU method B.13/14, OECD guideline 471 and EPA OPPTS 870.5100. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were tested in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). Two independent experiments were run (Initial Mutation Assay and Confirmatory Mutation Assay) up to limit or cytotoxic concentrations, with and without metabolic activation (S9 mix). All assays, including controls, were tested in triplicate.

Inhibitory effects of treatment were observed in all phases of the study. In the experiment I the inhibitory effect appeared in reduction of spontaneous rate compared to the solvent control values (Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537), and in background lawn reduction (Salmonella typhimurium TA 100, TA 1535 and TA 1537). In the experiment II revertant colony numbers compared to the solvent control plates as well as the background lawn development were reduced and small pinpoint colonies appeared in the investigated Salmonella typhimurium strains. Reduction of revertant colony numbers was pronounced at high concentration levels (5000.00-158.11 µg/plate) with metabolic activation, but observed down to lower concentrations in non-activated treatments. In test strains Salmonella typhimurium TA100, TA 1535 and TA1537 cytotoxic effects were observed even at lowest concentrations (5.00 µg/plate) and for TA 98 observed down to 15.81 µg/plate. No cytotoxic effects were observed in Escherichia coli WP2uvrA. In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. Cytotoxic effects were observed at concentrations of 50.00-5.00 µg/plate without metabolic activation. At the lower concentrations no cytotoxic effects were noted.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with SIKA Hardener LH 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.

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

 

In-vitro Mammalian Chromosome Aberration Test

SIKA Hardener LH was tested in a Chromosome Aberration Assay in CHO-KI cells according to EU method B.10, OECD guideline 473 and EPA OPPTS 870.5375. The test item was dissolved in Acetone and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A

without S9 mix: 4-h treatment, 20-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

with S9 mix: 4-h treatment, 20-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

Experiment B

without S9 mix: 20-h treatment, 28-h preparation interval

20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL and 150 µg/mL SIKA Hardener LH

with S9 mix: 4-h treatment, 28-h preparation interval

50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL and 600 µg/mL SIKA Hardener LH

The highest concentrations of test item SIKA Hardener LH (80, 150 µg/mL in the absence of S9 mix and 600 µg/mL in the presence of S9 mix) were cytotoxic and no viable cells remained. In Experiment A no significant increases in the number of cells showing structural chromosome aberrations were observed up to and including cytotoxic concentrations in the absence of metabolic activation. In the presence of metabolic activation the test item (200 µg/mL and 400 µg/mL) slightly increased the frequency of the cells with structural chromosome aberrations when compared to the concurrent control and when compared to historical control. This slightly higher frequency of the cells with structural chromosome aberrations was not biologically significant as it was noted at cytotoxic concentrations and was not observed in Experiment B.

In Experiment B no increase in the frequency of cells with structural chromosome aberrations (50 µg/mL, 100 µg/mL, 200 µg/mL and 400 µg/mL) was observed. When assessed without metabolic activation over a treatment period of 28 hours the frequency of the cells with structural chromosome aberrations was comparable to the concurrent control up to and including cytotoxic concentrations.

In Experiment B there were no significant increases in the number of cells showing structural chromosome aberrations without gaps, up to and including cytotoxic concentrations either in the absence or in the presence of metabolic activation.

There was no biologically relevant increase in the rate of polyploid and endoreduplicated metabphases in either experiment in the presence or in the absence of metabolic activation. 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.

In summary, SIKA Hardener LH tested both with and without metabolic activation did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells. Therefore, SIKA Hardener LH was considered not clastogenic.

 

In-vitro Mammalian Cell Gene Mutation Test

SIKA Hardener LH was tested in a Mammalian Gene Mutation Test in V79 cells. The test item was dissolved in N,N-Dimethylformamide and the following concentrations were selected on the basis of preliminary cytotoxicity investigations, without and with metabolic activation using S9 mix. Two independent experiments (both run in duplicate) with at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum toxicity (< 50 % survival):

Experiment 1

5-hour treatment period without and with S9 mix:

39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

Experiment 2

20-hour treatment period without S9 mix:

19.6, 39.1, 78.2 104.2, 130.2, 156.3 and 234.5 μg /mL

5-hour treatment period with S9 mix:

39.1, 78.2, 156.3, 312.5, 468.8, 625, and 937.5 μg/mL

In Experiment 1, there was a slightly higher mutation frequency at 39.1, 78.2, 312.5, 468.8, 625 and 937.5 μg/mL in the absence of metabolic activation and 39.1, 78.2, 156.3, 468.8, 625 and 937.5 μg/mL in the presence of metabolic activation compared to the concurrent control. These alterations were not biologically or statistically significant and no dose-response relationships were noted.

In Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted. The mutant frequency of the cells was slightly higher compared to the concurrent negative (solvent) control, but these alterations were not statistically significant when observed over a prolonged treatment period of 20 hours in the absence of metabolic activation. At all concentrations the mutant frequencies were above of negative control values when the test item was observed in the presence of metabolic activation, but these alterations were not toxicologically significant, further indicating that the findings in Experiment 1 were within the normal biological variation.

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 LH tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster lung cells. SIKA Hardener LH was not mutagenic in this in vitro mammalian cell gene mutation test performed with V79 cells.

Justification for classification or non-classification

Based on results of three different in vitro genetic toxicity studies, SIKA Hardener LH 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.