Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2007-10-16 to 2007-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000/32/EEC

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 1996/August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

614-657-1

Cas Number:

68609-08-5

Molecular formula:

C41H68N4O4

IUPAC Name:

Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

In the above experimental phases the examined concentration levels were different. The test item was dissolved in Dimethyl sulfoxide (DMSO).
In the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100-0.003 μg/plate with and without metabolic activation (± S9 Mix); in Escherichia coli WP2 uvrA: 3419-0.158 μg/plate (± S9 Mix).
In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10-0.003 μg/plate (–S9 Mix) and 100-0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1-0.158 μg/plate (± S9 Mix).

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine, NPD

Remarks:

TA 98 without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

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

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 Migrated to IUCLID6: 9AA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2uvrA without S9 Migrated to IUCLID6: MMS

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

S. typhimurium TA 100, TA 98, TA 1535, TA 1537; E. coli WP2uvrA with S9

Evaluation criteria:

The colony numbers for control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The mutation factor (MF) was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact, not rounded values were used for this calculation).

Statistics:

not applicable

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in plate incorporation tests (Initial Mutation Test, Complementary Plate Incorporation Test, Second Complementary Plate Incorporation Test) and in pre-incubation tests (Confirmatory Mutation Test and Complementary Pre-Incubation Test).

In general, the pre-incubation method is more sensitive than the plate incorporation method. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently). In the Complementary Pre-Incubation Test only the activated part (+S9 Mix) of assay was examined. The Complementary Pre-Incubation Test was performed with Salmonella typhimurium strains.
Following concentrations of Reaction Product of Bisphenol A (BADGE) with IPDA were tested in different experiments: in the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100-0.003 μg/plate without and with addition of metabolic activation (±S9 Mix), in Escherichia coli WP2 uvrA: 3419-0.158 μg/plate (± S9 Mix).

In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10-0.003 μg/plate (–S9 Mix) and 100-0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1-0.158 μg/plate (± S9 Mix).

In the Initial Mutation Test, in case of Salmonella typhimurium strains inhibition was observed in the examined concentration range (1-0.003 μg/plate) neither in presence, nor in absence of metabolic activation (S9 Mix).

The observed revertant colony numbers were slightly lower than the revertant colony numbers of the solvent control plates at different concentrations in case of Salmonella typhimurium TA 98, TA 1535 and TA 1537; however these variations were of the minor intensity and were considered to reflect the biological variability of the test.

In Escherichia coli WP2 uvrA the examined concentration range was higher (3419–1 μg/plate) than in the Salmonella typhimurium strains. In case of Escherichia coli WP2 uvrA no bacterial growth was observed in the concentration range of 3419-100 μg/plate without and in the range of 3419-341.9 μg/plate with addition of metabolic activation. The revertant colony numbers were reduced compared to the revertant colony numbers of the solvent control plates and the background lawn development was inhibited at the concentration of 34.19 μg/plate without, and at 100 μg/plate with addition of metabolic activation. The slightly lower revertant counts (compared to the solvent control plates) at the concentrations of 10 and 3.419 μg/plate (–S9 Mix), furthermore at 34.19 μg/plate (+S9 Mix) were evaluated as reflecting the biological variability in the test.

In the Complementary Plate Incorporation Test the examined concentration levels were 10, 3.162 and 1 μg/plate in the Salmonella typhimurium strains and 50; 15.81; 5; 1.581; 0.500 and 0.158 μg/plate in case of Escherichia coli WP2 uvrA. Unequivocally inhibitory effect (indicated by reduced revertant colony numbers compared to the solvent control plates and reduced background lawn development) of the test item was observed in all Salmonella typhimurium strains at the concentration of 10 μg/plate, and in the Escherichia coli WP2 uvrA at 50 μg/plate, without metabolic activation. Similarly to the Initial Mutation Test the slightly lower than the revertant colony numbers of the solvent control plates at different concentration levels in the examined strains were considered to reflect the biological variability of the test.

Since the cytotoxicity of the test item altered in presence of metabolic activation system (S9 Mix), a Second Complementary Plate Incorporation Test was carried out using the concentration levels of 100 and 31.62 μg/plate in Salmonella typhimurium strains and 500 and 158.1 μg/plate in Escherichia coli WP2 uvrA.
No bacterial growth was observed in the bacterial strains at the above concentration levels in absence of metabolic activation furthermore at 100 μg/plate (Salmonella typhimurium strains) and 500 and 158.1 μg/plate (Escherichia coli WP2 uvrA) in presence of metabolic activation. Furthermore reduced revertant colony number and background lawn development was observed in Salmonella typhimurium TA 98 and TA 1537 at the concentration of 31.62 μg/plate in presence of metabolic activation.

In the Confirmatory Mutation Test (Pre-Incubation Test) the examined concentration levels were: 10; 3.162; 1; 0.316; 0.1; 0.032; 0.01 and 0.003 μg/plate in the Salmonella typhimurium strains and 158.1; 50; 15.81; 5; 1.581; 0.500 and 0.158 μg/plate in case of Escherichia coli WP2 uvrA.

Strong inhibitory effects of the test item were observed in all examined strains, however in Salmonella typhimurium strains only the non-activated part (–S9 Mix) was inhibited. The signs of the inhibitory effects included reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance. No bacterial growth was observed in the Salmonella typhimurium TA 98, TA 1535, TA 1537 strains at 10 μg/plate (–S9 Mix), in Escherichia coli WP2 uvrA at 158.1 and 50 μg/plate (–S9 Mix). In Escherichia coli WP2 uvrA at 158.1 μg/plate (+S9 Mix) only one revertant colony was counted on the three parallels.
In the case of Salmonella typhimurium TA 1537 at 3.162 g/plate (–S9 Mix) the revertant colony numbers were increased, but reduced background lawn development was observed and the colonies were small, pinpoint colonies. The revertant colony count were lower than the revertant count of the solvent control plates in several cases, however they were in the historical control data range in all cases.

A Complementary Pre-Incubation Test was performed to reach the cytotoxic level in presence of metabolic activation (+S9 Mix) in case of Salmonella typhimurium strains. The examined concentration levels were 100 and 31.62 μg/plate. No bacterial growth was observed in Salmonella typhimurium TA 98, TA 1535, and TA 1537 at the concentration level of 100 μg/plate, and inhibition was observed (reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance) in all strains at 31.62 μg/plate, furthermore in Salmonella typhimurium TA 100 at 100 μg/plate.
In the consecutive mutation tests the inhibitory, cytotoxic effect of the test item independently of the examined strains (Salmonella typhimurium or Escherichia coli) appeared down to and including the concentration level of 3.162 μg/plate in absence and down to and including the concentration level of 31.62 μg/plate in presence of metabolic activation.
Positive and negative controls were run concurrently. 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.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

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

Executive summary:

Experimental Phases of the Study

The test item was examined in the following phases: in Plate Incorporation Tests (Initial Mutation Test, Complementary Plate Incorporation Test, and Second Complementary Plate Incorporation Test) and Pre-Incubation Tests (Confirmatory Mutation Test and Complementary Pre-Incubation Test).

Exposure Concentrations

In the above experimental phases the examined concentration levels were different. The test item was dissolved in Dimethyl sulfoxide (DMSO).

In the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100 - 0.003 μg/plate without and with addition of metabolic activation (± S9 Mix), in Escherichia coli WP2 uvrA: 3419 - 0.158 μg/plate (± S9 Mix). In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10 - 0.003 μg/plate (–S9 Mix) and 100 - 0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1 - 0.158 μg/plate (± S9 Mix).

Bacteria

Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 strains and Escherichia coli WP2 uvrA were used.

Results and Discussion:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in independent experiments, in plate incorporation tests and in a pre-incubation tests. Each assay (except the Complementary Pre-Incubation Test) was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

Inhibitory, cytotoxic effects of the test item were observed in the experiments in all examined bacterial strains and included reductions of spontaneous mutation rates, when compared to the solvent control values, reduced background lawn development and appearances of small pinpoint colonies. Furthermore no bacterial growth was observed for the strains at several concentration levels, with and without metabolic activation. Inhibition was stronger in the non-activated part (–S9 Mix) of the experiments.

No significant increases in revertant colony numbers of any of the five test strains were observed following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Further, there was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance in the performed experiments.

Positive and negative controls were run concurrently. 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.

Conclusion:

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