1,6-Hexanediamine, N1,N6-bis[2,2-dimethyl-3-(4-morpholinyl)propylidene]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for mutagenic activity in a bacterial reverse mutation assay according to EU Method B13/14 and OECD Guideline 471. The test item showed no mutagenic activity in the bacterial reverse mutation assay.

The test item was tested for clastogenic activity in mammalian cells according to OECD Guideline 473. The test item showed no clastogenic activity in this assay.

The test item was tested for its mutagenic activity in mammalian (CHO-K1) cells according to OECD Test Guideline 476 . The test item showed no mutagenic activity in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity S2A: “Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals” (1996) and S2B: Guidance on Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals “(1997)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S-9 mix

Test concentrations with justification for top dose:

15.8, 50, 158, 500, 1581, 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

S. typh. TA98, TA100, TA1535, TA1537 and E. coli with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

S. typh. TA98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typh. TA100 and TA1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

S. typh. TA1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli without metabolic activation

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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

Remarks on result:

other: all strains/cell types tested

In the Initial Mutation Test significantly higher revertant colony counts than the revertant colony numbers of the vehicle control plates (but within the actual historical control data range) were observed in S. typhimurium TA1535, at the concentration of 5000 μg/plate, with addition of metabolic activation (+S9 Mix). The MR value was 2.35. This high value was unique and was not accompanied by additional dose-relationship.

In the Initial Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates; furthermore the obtained higher revertant counts were above the corresponding historical control data range in the case of S. typhimurium TA1537 at the concentrations of 500, 50 and 15.8 μg/plate, without metabolic activation (-S9 Mix).

Higher revertant counts were obtained, however within the historical control data range in S. typhimurium TA98, at the concentrations of 5000 and 1581 μg/plate (-S9 Mix) and in the whole concentration range (5000 - 15.8 μg/plate (+S9 Mix), in S. typhimurium TA1535, at the concentrations of 1581, 50 and 15.8 μg/plate (+S9 Mix), moreover in TA1537 at 15.8 μg/plate (+S9 Mix). The higher revertant colony counts remained the threshold for being positive in all cases.

The revertant colony numbers remained in the vehicle control data range, showed no increases or decreases, but were slightly above the corresponding historical control data ranges in S. typhimurium TA100 at the concentrations of 5000, 158 and 50 μg/plate (-S9 Mix) and at 500 and 15.8 μg/plate (+S9 Mix); in TA1537 at the concentration of 5000 μg/plate, without metabolic activation (-S9 Mix).

In the Initial Mutation Test lower revertant colony counts (than the revertant colony counts of the vehicle control) were observed in the corresponding historical control data ranges in S. typhimurium TA1535 in the concentration range of 5000-158 μg/plate and additionally at 15.8 μg/plate, without metabolic activation (-S9 Mix) and in TA1537 at the concentration of 158 μg/plate (+S9 Mix).

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

In the Confirmatory Mutation Test, following the pre-incubation procedure inhibitory effect of the test item was observed in all examined bacterial strains at the highest concentration level of 5000 μg/plate, with and without metabolic activation (±S9 Mix). The low revertant colony counts were below the corresponding historical control data ranges, additionally reduced or slightly reduced background lawn development was observed at 5000 μg/plate in S. typhimurium TA98, (±S9 Mix), in TA1537 and E. coli WP2 uvrA (-S9 Mix) and in TA100 (+S9 Mix). The lower revertant colony counts (than the revertant colony counts of the vehicle control plates) remained in the historical control data ranges, but the affected background lawn development indicated the inhibitory effect of the test item at 5000 μg/plate in S. typhimurium TA1535 (±S9 Mix), in TA1537 and E. coli WP2 uvrA (+S9 Mix). No revertant growth and reduced background lawn development was obtained in S. typhimurium TA100, at 5000 μg/plate (-S9 Mix).

The revertant colony numbers were slightly higher than the revertant colony numbers of the vehicle control plates in S. typhimurium TA98, at the concentrations of 1581 and 500 μg/plate (±S9 Mix), in TA1535, at 158 μg/plate (-S9 Mix), and in E. coli WP2 uvrA at the concentrations of 1581, 500, 158 and 15.8 μg/plate (+S9 Mix). These revertant colony number increases remained in the corresponding historical control data ranges. In the Confirmatory Mutation Test slightly lower revertant colony counts (compared to the vehicle control) in the historical control data ranges were observed in S. typhimurium TA98 at the concentration of 15.8 μg/plate (-S9 Mix), in TA1535 at 15.8 μg/plate (+S9 Mix), in TA1537 at 1581 μg/plate (-S9 Mix) and at 158 and 15.8 μg/plate (+S9 Mix). These lower revertant colony counts were within the biological variability of the applied test system.

The revertant colony numbers of vehicle control (DMSO) plates with and without S9 Mix were within the corresponding historical control data ranges in both experiments (Initial and Confirmatory Mutation Test)*.

* With exception of the slightly lower revertant colony numbers of the S. typhimurium TA98.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The revertant colony numbers of the untreated and distilled water control plates in the different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Sika Hardener MH at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusions:

The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Sika Hardener MH was considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item SIKA Hardener MH was tested for mutagenic activity in a bacterial reverse mutation assay according to EU Method B13/14 and OECD Guideline 471.

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Sika Hardener MH in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases there were at least five analysable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Sika Hardener MH at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values and/or revertant colony numbers above the actual historical control data ranges were observed in both independently performed main experiments.

There was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

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

The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Sika Hardener MH 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:

2019-06-19 to 2019-07-29

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I1900071-19158-AKM
- Purity: 99.8 % (aldimine group contents via acid titration)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 chinese hamster cells (male);
- Supplier: ECACC (European Collection of Cell Cultures)
- Suitability of cells: according to the guideline
- Normal cell cycle time (negative control): doubling time 12-14 h

For cell lines:
- Absence of Mycoplasma contamination: yes
- doubling time: 12-14 h
- Modal number of chromosomes: 2n=22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media:
The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Based on a cytotoxicity pre-test the following concentrations were chosen for the main test:

Experiment A with 3/20 h treatment/sampling time: without and with S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/20 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item
Experiment B with 20/28 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item
Experiment B with 3/28 h treatment/sampling time: with S9 mix: 125, 250, 500 and 750 μg/mL test item

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: suitable solubility of the test item and for application in the cell culture used; recommended by the respective guidelines

- Justification for percentage of solvent in the final culture medium: as recommended by the guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: dublicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 105 cells per culture
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: not applicable
- Exposure duration/duration of treatment: 20 hours (-S9) and 3 hours (+S9)
- Harvest time after the end of treatment (sampling/recovery times): 20 and 28 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: colchicine, 0.2 µg/mL, 2.5 h prior to harvesting
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide)
- Criteria for scoring chromosome aberrations: The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)

Rationale for test conditions:

According to the respective guidelines.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: ok
- Data on osmolality: ok
- Possibility of evaporation from medium: no
- Water solubility: well
- Precipitation and time of the determination: no
- Other confounding effects: none

Table 1 Mean percentage of cells with structural chromosome aberrations: Experiment A

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150 cells
				incl. gaps	excl. gaps
Solvent control (DMSO)	-	3 h	20 h	7	3
Test item
125µg/mL	-	3 h	20 h	7	3
250µg/mL	-	3 h	20 h	9	3
500µg/mL	-	3 h	20 h	8	4
750µg/mL	-	3 h	20 h	8	4
Pos. Control (EMS)	-	3 h	20 h	39**	34**
Solvent control (DMSO)	+	3 h	20 h	8	3
Test item
125µg/mL	+	3 h	20 h	7	3
250µg/mL	+	3 h	20 h	8	3
500µg/mL	+	3 h	20 h	9	3
750µg/mL	+	3 h	20 h	10	4
Pos. Control (Cycl.)	+	3 h	20 h	47**	41**

Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL)

Positive control (+S9): Cyclophosphamide (5.0 µg/mL)

**: p<0.01

Table 2 Mean percentage of cells with structural chromosome aberrations: Experiment B

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Solvent control (DMSO)	-	20 h	20 h	7	3
Test item
15.7 µg/mL	-	20 h	20 h	7	3
31.3µg/mL	-	20 h	20 h	7	3
62.5µg/mL	-	20 h	20 h	7	3
125µg/mL	-	20 h	20 h	9	4
Pos. Control (EMS)	-	20 h	20 h	43**	39**
Solvent control (DMSO)	-	20 h	28 h	6	3
Test item
15.7 µg/mL	-	20 h	28 h	6	3
31.3µg/mL	-	20 h	28 h	7	3
62.5µg/mL	-	20 h	28 h	7	3
125µg/mL	-	20 h	28 h	6	3
Pos. Control (EMS)	-	20 h	28 h	42**	39**
Solvent control (DMSO)	+	3 h	28 h	7	3
Test item
125µg/mL	+	3 h	28 h	8	3
250µg/mL	+	3 h	28 h	7	3
500µg/mL	+	3 h	28 h	7	2
750µg/mL	+	3 h	28 h	9	3
Pos. Control (Cycl.)	+	3 h	28 h	45**	42**

Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL)

Positive control (+S9): Cyclophosphamide (5.0 µg/mL)

**: p<0.01

Conclusions:

In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Executive summary:

The test item, dissolved in DMSO, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

- Experiment A with 3/20 h treatment/sampling time: without and with S9 mix: 125, 250, 500 and 750 μg/mL test item

- Experiment B with 20/20 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item

- Experiment B with 20/28 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item

- Experiment B with 3/28 h treatment/sampling time: with S9 mix: 125, 250, 500 and 750 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. There were no of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the laboratory historical control data. The concurrent positive controls ethyl methanesulphonate (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 and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic 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:

2019-06-25 to 2019-09-23

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 using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I1900071-19158-AKM
- Purity: 99.8 % (aldimine group contents via acid titration)

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)

MEDIA USED
- 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 %).
During the 5 hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL of 6-thioguanine (6-TG)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Without S9: 250; 500; 750; 1000; 1250; 1500 µg/mL
With S9: 7.5; 62.5; 125; 250; 350; 500; 750 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: in accordance with guideline, solubility of test item and suitability with cell culture

- Justification for percentage of solvent in the final culture medium: according to the guideline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10^6 cells per dish

DURATION
- Preincubation period: 24 h
- Exposure duration: 5 h
- Expression time: 19 h
- Selection time: 8 d

SELECTION AGENT: hypoxanthine Ham's (F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Rationale for test conditions:

In accordance with guideline.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, 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:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: unremarkable
- Data on osmolality: unremarkable
- Possibility of evaporation from medium: no
- Water solubility: well
- Precipitation and time of the determination: no
- Other confounding effects: none

Table 1 Results summary (5 hour Treatment without S9) run 1

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control (DMSO) a	200.3	±	2.08	100	100	6	101	5.94
Pos. control (EMS 1.0µL/mL) a	55.7	±	2.08	28	66	1011	67	1508.96**
TEST ITEM
250g/mL a	194,0	±	1,00	97	100	6	101	5.94
500g/mL a	176,7	±	2,08	88	99	6	100	6.00
750g/mL a	160,7	±	1,15	80	99	8	100	8.00
1000g/mL a	129,7	±	1,53	65	98	5	99	5.05
1250g/mL a	91,7	±	1,53	46	99	6	100	6.00
1500g/mL a	34,0	±	1,00	17	97	8	99	8.08

a = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 2 Results summary (5 hour Treatment without S9) run 2

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control b	201.7	±	0.58	100	100	6	101	5.94
Pos. control (EMS 1.0µL/mL) b	55.0	±	2.0	27	67	1005	68	1477.94**
TEST ITEM
250g/mL b	193,7	±	1,53	96	99	5	100	5.00
500g/mL b	176,3	±	1,15	87	99	5	100	5.00
750g/mL b	162,0	±	1,00	80	99	8	100	8.00
1000g/mL b	130,0	±	1,00	64	98	6	99	6.06
1250g/mL b	92,3	±	1,53	46	99	6	100	6.00
1500g/mL b	34,0	±	1,00	17	98	7	99	7.07

b = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control

 

Table 3 Results summary (5 hour Treatment without S9) run 3

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control c	201.0	±	2.65	100	100	7	101	6.93
Pos. control (EMS 1.0µL/mL) c	53.3	±	1.53	27	70	1021	71	1438.03**
TEST ITEM
250g/mL c	196,3	±	2,08	98	100	6	101	5.94
500g/mL c	174,0	±	3,61	87	99	5	99	5.05
750g/mL c	167,0	±	2,65	83	100	6	100	6.00
1000g/mL c	137,3	±	3,21	68	98	6	99	6.06
1250g/mL c	96,7	±	0,58	48	98	6	99	6.06
1500g/mL c	35,3	±	0,58	18	98	7	99	7.07

 

c = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 4 Results summary (5 hour Treatment without S9) run 4

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control d	202.7	±	2.52	100	100	6	101	5.94
Pos. control (EMS 1.0µL/mL) d	52.7	±	2.52	26	71	1035	71	1457.75**
TEST ITEM
250g/mL d	196,0	±	1,00	97	100	6	100	6.00
500g/mL d	174,3	±	3,06	86	99	5	100	5.00
750g/mL d	168,7	±	2,31	83	100	7	101	6.93
1000g/mL d	132,0	±	1,00	65	99	5	100	5.00
1250g/mL d	93,3	±	1,53	46	99	6	99	6.06
1500g/mL d	33,7	±	2,08	17	99	5	99	5.05

d = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 5 Results summary (5 hour Treatment with S9) run 1

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control a	199.7	±	1.53	100	100	6	101	5.94
Pos. control (DMBA20 µg/mL) a	122.3	±	1.53	61	72	516	72	716.67**
TEST ITEM
62.5g/mL a	193,7	±	1,53	97	99	7	100	7.00
125g/mL a	172,3	±	3,21	86	98	8	99	8.08
250g/mL a	161,3	±	1,53	81	98	8	99	8.08
350g/mL a	135,7	±	3,21	68	99	6	99	6.06
500g/mL a	82,3	±	2,08	41	98	8	99	8.08
750g/mL a	26,0	±	1,00	13	98	6	98	6.12

a = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control

 

 

Table 6 Results summary (5 hour Treatment with S9) run 2

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control b	201.0	±	1.0	100	100	7	101	6.93
Pos. control (DMBA20 µg/mL) b	123.3	±	1.53	61	71	530	72	736.11**
TEST ITEM
62.5g/mL b	194,7	±	2,08	97	100	5	100	5.00
125g/mL b	171,7	±	2,89	85	99	7	99	7.07
250g/mL b	161,0	±	1,00	80	99	8	99	8.08
350g/mL b	134,0	±	1,00	67	100	6	100	6.00
500g/mL b	80,7	±	1,53	40	99	5	99	5.05
750g/mL b	27,0	±	1,00	13	99	8	99	8.08

b = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control

 

 

 

Table 7 Results summary (5 hour Treatment with S9) run 3

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control c	199.7	±	0.58	100	100	7	100	7.00
Pos. control (DMBA20 µg/mL) c	120.7	±	1.53	60	74	521	74	704.05**
TEST ITEM
62.5g/mL c	193,3	±	1,15	97	97	8	98	8.16
125g/mL c	174,7	±	1,53	87	99	7	100	7.00
250g/mL c	161,3	±	0,58	81	98	7	99	7.07
350g/mL c	126,0	±	2,65	63	98	8	98	8.16
500g/mL c	81,7	±	0,58	41	98	6	98	6.12
750g/mL c	27,0	±	1,00	14	97	7	98	7.14

c = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control

 

 

 

Table 8 Results summary (5 hour Treatment with S9) run 4

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL
Solvent control d	200.7	±	0.58	100	100	8	101	7.92
Pos. control (DMBA20 µg/mL) d	121.7	±	1.53	61	73	520	73	712.33**
TEST ITEM
62.5g/mL d	193,7	±	1,15	97	98	8	99	8.08
125g/mL d	175,0	±	1,73	87	99	6	100	6.00
250g/mL d	162,3	±	0,58	81	99	6	100	6.00
350g/mL d	126,0	±	1,00	63	98	8	98	8.16
500g/mL d	80,7	±	1,15	40	99	7	99	7.07
750g/mL d	27,3	±	0,58	14	98	7	99	7.07

d = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control

 

Conclusions:

The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency in an OECD 476 compliant study. Thus, the test item was not mutagenic under the conditions of this study.

Executive summary:

The test item, dissolved in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

 

5-hour treatment period without S9-mix:

250, 500, 750, 1000, 1250 and 1500 µg/mL

5-hour treatment period with S9-mix:

62.5, 125, 250, 350, 500 and 750 µg/mL

 

In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. In the absence and presence of metabolic activation clear cytotoxicity (survival between 13-18 %) of the test item was observed at the highest concentration applied (1500 µg/mL in the absence and 750 µg/mL in the presence of S9 mix). There were no relevant changes osmolality after treatment with the test item. The pH values of the solutions containing the test item were slightly above the control value. Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). 

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471 - Ames-Test:

The test item was tested for mutagenic activity in a bacterial reverse mutation assay according to EU Method B13/14 and OECD Guideline 471.

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Sika Hardener MH in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases there were at least five analysable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Sika Hardener MH at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values and/or revertant colony numbers above the actual historical control data ranges were observed in both independently performed main experiments.

There was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

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

The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, Sika Hardener MH was considered non-mutagenic in this bacterial reverse mutation assay.

OECD 473 - Chromosome Aberration Assay

The test item, dissolved in DMSO, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

- Experiment A with 3/20 h treatment/sampling time: without and with S9 mix: 125, 250, 500 and 750 μg/mL test item

- Experiment B with 20/20 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item

- Experiment B with 20/28 h treatment/sampling time: without S9 mix: 15.7, 31.3, 62.5 and 125 μg/mL test item

- Experiment B with 3/28 h treatment/sampling time: with S9 mix: 125, 250, 500 and 750 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations compared to concurrent controls or in comparison with the range of historical controls were observed, neither in the absence nor in the presence of metabolic activation. There were no of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the laboratory historical control data. The concurrent positive controls ethyl methanesulphonate (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 and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

OECD 476 - Gene mutation in mammalian cells (HPRT Assay)

The test item, dissolved in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

 

5-hour treatment period without S9-mix:

250, 500, 750, 1000, 1250 and 1500 µg/mL

5-hour treatment period with S9-mix:

62.5, 125, 250, 350, 500 and 750 µg/mL

 

In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. In the absence and presence of metabolic activation clear cytotoxicity (survival between 13-18 %) of the test item was observed at the highest concentration applied (1500 µg/mL in the absence and 750 µg/mL in the presence of S9 mix). There were no relevant changes osmolality after treatment with the test item. The pH values of the solutions containing the test item were slightly above the control value. Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). 

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

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

 

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 for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the twelfth time in Regulation (EU) No. 2019/521.