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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2008-08-08 to 2009-03-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of benzene-1,3-diyldimethanamine and 3-aminomethyl-3,5,5-trimethylcyclohexanamine with oligomerisation products of 4,4'-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane
EC Number:
700-128-3
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Reaction products of benzene-1,3-diyldimethanamine and 3-aminomethyl-3,5,5-trimethylcyclohexanamine with oligomerisation products of 4,4'-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane
Details on test material:
NA

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 frame shift (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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
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:
The test item was dissolved in Dimethyl sulfoxide (DMSO). In the Initial Mutation Test and Confirmatory Initial Mutation Test the examined concentrations were: 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 µg/plate for Salmonella tester strains; 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 µg/plate for Escherichia coli tester strain. This series was completed in the Complementary Initial Mutation Test with additional concentration levels of 500, 158.1, 50 µg/plate for S. typhimurium TA1535 strain; 158.1 and 50 µg/plate for S. typhimurium TA1537 strain; 0.5, 0.1581 and 0.05 μg/plate for E. coli WP2 tester strain. In the Confirmatory Mutation Test the examined concentrations were: 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 µg/plate for Salmonella tester strains; 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 µg/plate for Escherichia coli tester strain. This series was completed in the Complementary Confirmatory Mutation Test with additional concentration levels of 158.1, 50, 15.81 µg/plate for Salmonella tester strains; 500, 158.1, 50 µg/plate for E.coli tester strain.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controlsopen allclose all
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
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
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
W. coli WP2uvrA, without S9
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 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).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
50 µg/plate without S9; 50; 158.1; 500 µg/plate with S9
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
Remarks:
50 µg/plate without S9; 50; 158.1; 500 µg/plate with S9
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
Remarks:
50 µg/plate without S9; 50; 158.1; 500 µg/plate with S9
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
Remarks:
50 µg/plate without S9; 50; 158.1; 500 µg/plate with S9
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:
cytotoxicity
Remarks:
500; 158.1 µg/plate without S9; 500 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 TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in four independent experiments, in two plate incorporation test (Experiment I, Initial Mutation Test and Experiment III, Complementary Initial Mutation Test) and in two pre-incubation tests (Experiment II, Confirmatory Mutation Test and Experiment IV, Complementary Confirmatory Mutation Test). In general, the pre-incubation method is more sensitive than the plate incorporation assay. Each assay was conducted with and without metabolic activation system (± S9 Mix). The concentrations, including the untreated, vehicle and positive controls, were tested in triplicate.

Following concentrations of the test item were tested in Experiment I (the concentrations were chosen based on the results of the Pre-Experiment): 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate for Salmonella typhimurium tester strains; 500; 158.1; 50; 15.81; 5; 1.581 and 0.5 µg/plate for Escherichia coli WP2 uvrA bacterial strain. Additional concentration levels were investigated in the complementary Experiment III based on the results of the Experiment I. These additional concentration levels were: 500; 158.1 and 50 µg/plate for TA1535 strain with metabolic activation system (+S9 Mix), 158.1 and 50 µg/plate for TA1537 strain with metabolic activation system (+S9 Mix), 0.5; 0.1581 and 0.05 µg/plate in E. coli WP2 strain without metabolic activation system (-S9 Mix).

Using the plate incorporation method (Initial Mutation Test and Complementary Initial Mutation Test) the highest revertant numbers compared to the revertant numbers of the solvent control plates were observed in the Initial Mutation Test in TA1537 tester strain at 0.05 µg/plate concentration without metabolic activation. The mutation factor value was 1.93 in this case. This number was not dose-related, below the biological relevant threshold value and within the historical control range.

Sporadically lower revertant colony numbers compared to the solvent control were observed, but all of those revertant numbers were in the historical control range.

Strong cytotoxic, inhibitory effect of the test item was observed in plate in-corporation experiments. No revertant colonies grew and the absence of background lawn development or reduced background lawn was detected in TA98 tester strain at 50 µg/plate without metabolic activation; in TA1535 tester strain at 50 µg/plate without metabolic activation and at 500 and 158.1 µg/plate with metabolic activation; in TA1537 tester strain at 50 µg/plate without metabolic activation and at 158.1 and 50 µg/plate with metabolic activation; in E.coli tester strain at 500 and 158.1 µg/plate without metabolic activation and at 500 µg/plate with metabolic activation. Reduced background lawn development and reduced revertant rates compared to the solvent control were observed in TA98 tester strain at 15.81 µg/plate without metabolic activation and 50 µg/plate with metabolic activation, in TA100 tester strain at 50 µg/plate concentration without metabolic activation; in TA1537 tester strain at 15.81 µg/plate concentration without metabolic activation; in E.coli tester strain 50 µg/plate without metabolic activation and at 158.1 µg/plate concentration with metabolic activation. Slightly reduced background lawn development and lower number of revertant colonies compared to the solvent control were observed in TA100 bacterial strain at 15.81 µg/plate without metabolic activation and 50 µg/plate with metabolic activation; in TA1535 tester strain at 15.81 µg/plate without metabolic activation system. Slightly reduced background lawn development and appearance of pinpoint colonies were detected in TA1537 bacterial strain at the concentration of 50 µg/plate with metabolic activation.

Following concentrations of the test item were tested in Experiment II: 15.81; 5; 1.581; 0.5; 0.1581; 0.05 and 0.01581 µg/plate for Salmonella thyphimurium tester strains; 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate for E.coli WP2 uvrA bacterial strain. Additional concentration levels were investigated in the complementary Experiment IV based on the results of the Experiment II. These additional concentration levels were: 158.1, 50 and 15.81 µg/plate for Salmonella typhimurium tester strains with metabolic activation system (+S9 Mix), 500; 158.1 and 50 µg/plate for E. coli WP2 strain with metabolic activation system (+S9 Mix).

Using the pre-incubation method (Confirmatory Mutation Test and Complementary Confirmatory Mutation Test) the highest number of revertant colonies compared to the number of revertant colonies on the solvent control plates was observed in Salmonella typhimurium TA1535 tester strain at 1.581 μg/plate concentration with metabolic activation. The mutation factor value was 1.57. However, the revertant colony number remained in the historical control range, there was no dose-dependence and this value is well below the biological relevant threshold value.
Sporadically lower number of revertant colonies compared to the solvent control was observed in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, however those values remained in the historical control range.

A strong inhibitory, cytotoxic effect of the test item was observed by using the pre-incubation method. No revertant colonies grew and the absence of background lawn development was detected in TA1537 tester strain at 15.81 µg/plate without metabolic activation and in all Salmonella strains at 158.1 µg/plate with metabolic activation. Absence of background lawn or reduced background lawn development was detected in E.coli WP2 tester strain at 500 and 158.1 µg/plate with metabolic activation, and beside of the inhibited background lawn development no revertant colonies grew on some plates. Absent or reduced background lawn development and reduced revertant rates compared to the solvent control were observed in TA98, TA100 and TA1535 tester strains at 15.81 µg/plate concentration without metabolic activation; in TA1535 and TA1537 tester strains at 5 µg/plate concentration without metabolic activation; in all Salmonella strains at 50 µg/plate concentration with metabolic activation; in E.coli tester strain at 50 µg/plate without metabolic activation. Slightly reduced background lawn development and lower number of revertant colonies compared to the solvent control were observed in TA98 bacterial strain at 5 µg/plate without metabolic activation; in E.coli tester strain in the Confirmatory Mutation Test at 50 µg/plate with metabolic activation.

Positive and negative controls were run concurrently in each experiment. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. The viability of bacterial cells was checked by a plating experiment in all tests.

Applicant's summary and conclusion

Conclusions:
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:

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 four independent experiments, in two plate incorporation tests (Experiment I, Initial Mutation Test and Experiment III, Complementary Initial Mutation Test) and in two pre-incubation tests (Experiment II, Confirmatory Mutation Test and Experiment IV, Complementary Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including untreated controls, vehicle controls and positive controls, were tested in triplicate. The test item was dissolved in Dimethyl sulfoxide (DMSO). In the Initial Mutation Test and Confirmatory Initial Mutation Test the examined concentrations were: 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 µg/plate for Salmonella tester strains; 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 µg/plate for Escherichia coli tester strain. This series was completed in the Complementary Initial Mutation Test with additional concentration levels of 500, 158.1, 50 µg/plate for S. typhimurium TA1535 strain; 158.1 and 50 µg/plate for S. typhimurium TA1537 strain; 0.5, 0.1581 and 0.05 μg/plate for E. coli WP2 tester strain. In the Confirmatory Mutation Test the examined concentrations were: 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 µg/plate for Salmonella tester strains; 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 µg/plate for Escherichia coli tester strain. This series was completed in the Complementary Confirmatory Mutation Test with additional concentration levels of 158.1, 50, 15.81 µg/plate for Salmonella tester strains; 500, 158.1, 50 µg/plate for E.coli tester strain.

No substantial increases were observed in revertant colony numbers of any of the five test strains, following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the Initial and Confirmatory Mutation Tests.

The observed higher revertant colony numbers compared to the solvent control plates were mostly of minor intensity, did not follow a dose-response relationship, they were below the biological relevant threshold value and in the historical control range, so they were considered as reflecting the biological variability of the test.

Lower number of revertant colonies and reduced revertant rates compared to the solvent control plates were observed in some cases, but all of those values remained in the historical control range

Strong inhibitory, cytotoxic effects of the test item were observed in the experiments using both the plate incorporation and pre-incubation method. Absent, reduced or slightly reduced background lawn development was observed, and besides the inhibited background lawn development the number of revertant colonies (compared to the revertant colony numbers of the solvent control plates) was also reduced. The appearance of pinpoint colonies was detected on some plates.

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. 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.

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