Reaction products of m-phenylenebis(methylamine) 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 2008-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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)

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 m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

641-048-8

Cas Number:

110839-13-9

Molecular formula:

C37H48N4O4

IUPAC Name:

Reaction products of m-phenylenebis(methylamine) 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 200 - 0.002 μg/plate with (+S9 Mix), and 20 - 0.002 without metabolic activation (–S9 Mix); in Escherichia coli WP2 uvrA: 3419 - 0.2 μg/plate (±S9 Mix).
In the Pre-Incubation Tests: in the Salmonella typhimurium strains 20 - 0.006 μg/plate (–S9 Mix) and 200 - 0.006 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 200 - 0.063 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 with S 9, and E. coli WP2uvrA without 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).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 6.32 ug/plate without S9; <= 63.24 ug/plate with S9;

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 6.32 ug/plate without S9; <= 63.24 ug/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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 6.32 ug/plate without S9; <= 63.24 ug/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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 6.32 ug/plate without S9; <= 63.24 ug/plate with S9;

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 6.32 ug/plate without S9; <=63.24 ug/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 Second Complementary Plate Incorporation Test and Complementary Pre-Incubation Test only the activated part (+S9 Mix) of assay was examined. These tests were performed with Salmonella typhimurium strains.

Following concentrations of the test item were tested in different experiments: in the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 200 - 0.002 μg/plate with metabolic activation (+S9 Mix), and 20 - 0.002 without metabolic activation (–S9 Mix); in Escherichia coli WP2 uvrA: 3419 - 0.200 μg/plate (±S9 Mix).

In the Pre-Incubation Tests: in the Salmonella typhimurium strains 20 - 0.006 μg/plate (–S9 Mix) and 200 - 0.006 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 200 - 0.063 μg/plate (±S9 Mix).

In the Initial Mutation Test, in case of Salmonella typhimurium strains no inhibition was observed in the examined concentration range (2 - 0.002 μg/plate) either in presence, or 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 [at the concentration of 341.9 μg/plate 4 revertant colonies were counted on three plates (+S9 Mix)]. The revertant colony numbers were reduced compared to the revertant colony numbers of the solvent control plates and the background lawn development was slightly inhibited at the concentration of 100 μg/plate with addition of metabolic activation. The slightly lower revertant counts (compared to the solvent control plates) in the concentration range of 34.19-1.000 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 20, 6.32 and 2 μg/plate in the Salmonella typhimurium strains and 200; 63.24; 20; 6.32; 2; 0.632 and 0.200 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 or slightly background lawn development) of the test item was observed in all Salmonella typhimurium strains at the concentration of 20 μg/plate, and additionally in Salmonella typhimurium TA 100 at 6.32, without metabolic activation.
In case of Escherichia coli WP2 uvrA inhibition was observed at 200 μg/plate (±S9 Mix) and at 63.24 μg/plate (–S9 Mix). In Escherichia coli WP2 uvrA no bacterial growth was observed at 200 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. In the Second Complementary Plate Incorporation Test Salmonella typhimurium strains were examined at the concentration levels of 200 and 63.24 μg/plate. In this experiment only the activated part (+S9 Mix) of the assay was tested.
No bacterial growth was detected in the four Salmonella typhimurium strains at 200 μg/plate furthermore in TA 98 and TA 1537 at 63.24 μg/plate (in TA 98 1-1 revertant colony was counted on the plates). Reduced revertant colony numbers and background lawn development was observed in Salmonella typhimurium TA 100 and TA 1535 at the concentration of 63.24 μg/plate.

In the Confirmatory Mutation Test (Pre-Incubation Test) the examined concentration levels were: 20; 6.32; 2; 0.632; 0.2; 0.063; 0.02 and 0.006 μg/plate in the Salmonella typhimurium strains and 200; 63.24; 20; 6.32; 2; 0.632; 0.200 and 0.063 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. The above signs were observed in Salmonella typhimurium TA 98 and TA 1537 at 6.32 µg/plate (–S9 Mix). No bacterial growth was observed in the Salmonella typhimurium TA 98, TA 1535, TA 1537 strains at 20 μg/plate (–S9 Mix), in Escherichia coli WP2 uvrA at 200 μg/plate (–S9 Mix).
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 200 and 63.24 μg/plate. No bacterial growth was observed in all strains at the concentration level of 200 μg/plate, and in TA 98 and TA 1537 at 63.24 μg/plate. Further, inhibition was observed (reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance) in TA 100 and TA 1535 at 63.24 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 6.32 μg/plate in absence and down to and including the concentration level of 63.24 μg/plate in presence of metabolic activation.

In the Confirmatory Mutation Test (Pre-Incubation Test) higher revertant rates were observed in case of Salmonella typhimurium TA 98 at 2 μg/plate (MF=1.81), without metabolic activation and at 20 μg/plate (MF=2.49), with metabolic activation. There were no additional dose-dependent relationship and these values were well below the biological threshold value.

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.

Applicant's summary and conclusion

Conclusions:

The test item was tested for mutagenic properties in a Reverse Mutation Assay using bacteria. 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 200 - 0.002 μg/plate with (+S9 Mix), and 20 - 0.002 without metabolic activation (–S9 Mix); in Escherichia coli WP2 uvrA: 3419 - 0.2 μg/plate (±S9 Mix).

In the Pre-Incubation Tests: in the Salmonella typhimurium strains 20 - 0.006 μg/plate (–S9 Mix) and 200 - 0.006 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 200 - 0.063 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

The above five bacterial strains 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 Second Complementary Plate Incorporation Test and 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, (±S9 Mix). 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.