Bis(p-methoxyphenyl)iodonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The reported data of a mutagenicity assay according to OECD TG 471 show that under the experimental conditions applied, the test item induce gene mutations by frameshifts in the genome of the investigated Salmonella typhimurium TA1537 strain in the absence and presence of exogenous metabolic activation.

In conclusion, the test item DIMETHOXYDIPHENYL-IODONIUMBROMID has mutagenic activity on the applied Salmonella typhimurium TA1537 under the test conditions used in this study.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2019 - 28 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal enzyme activation mixture (rat liver extract, S9 fraction).

Test concentrations with justification for top dose:

5 to 5000 μg/plate

Vehicle / solvent:

In the study two vehicle control groups were used according to the solubility of the test item and the solubility of positive control reference items.
Dimethyl sulfoxide (DMSO) for Test Item, NPD, 9AA and 2AA;
Ultrapure water for SAZ and MMS.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD; 2-aminoanthracene, 2AA;

Details on test system and experimental conditions:

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 1.38 x 10^9 cells per mL.
The plates were incubated at 37 °C for 48 hours and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment. Three plates were used per concentration and per strain for the test substance; 6 for the negative control.

Evaluation criteria:

The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and mutation rates were calculated.
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 Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item 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:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Species / strain:

S. typhimurium TA 100

Genotoxicity:

ambiguous

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the performed experiments inhibitory, cytotoxic effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by decreased revertant colony counts (absent revertants or revertants below the corresponding historical control data ranges and/or corresponding vehicle) and/or affected colony development (pinpoint colonies) and/or affected background lawn development (reduced or slightly reduced background lawn). The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate (following the plate incorporation procedure in S. typhimurium TA1535 and TA1537; following the pre-incubation procedure in all S. typhimurium strains).
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and all of the number of revertants fell in the corresponding historical control ranges (or even were above the historical control data range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.
In the initial mutation test, following the plate incorporation procedure, positive results, significant, biologically relevant, dose-related changed revertant colony number increases, revertant colony numbers above the vehicle control data and above the historical control data ranges were obtained in S. typhimurium TA1537 strain in the absence and presence of exogenous metabolic activation (±S9). The biologically relevant increases were adequately confirmed in subsequent experiments. Furthermore, equivocal, borderline positive results, biological relevant revertant colony number increases were noticed in the confirmatory mutation test (pre-incubation test) in E. coli WP2 uvrA, in the absence of exogenous metabolic activation (-S9). These latter increases were not repeated, adequately confirmed, the higher revertant colony numbers remained below the relevant genotoxicological threshold for being positive in the subsequent experiment (repeated pre-incubation test).

Remarks on result:

other: all strains/cell types tested

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induce gene mutations by frameshifts in the genome of the investigated Salmonella typhimurium TA1537 strain in the absence and presence of exogenous metabolic activation.
In conclusion, the test item DIMETHOXYDIPHENYL-IODONIUMBROMID has mutagenic activity on the applied Salmonella typhimurium TA1537 under the test conditions used in this study.

Executive summary:

The test item DIMETHOXYDIPHENYL-IODONIUMBROMID was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The initial and confirmatory mutation experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (±S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. In this study additional (partial), or repeated tests were performed also performed investigating affected, representative strains, only.

For scientific and unforeseen technical reasons, beside the usual procedures (preliminary solubility test, concentration range finding test, initial mutation test and confirmatory mutation test) this study included an additional (partial) plate incorporation test with E. coli WP2 uvrA, an additional (partial) plate incorporation test with S. typhimurium TA1537 and a repeated pre-incubation test with E. coli WP2 uvrA1

Furthermore, the evaluation of the confirmatory mutation test in the case of Salmonella typhimurium strains was not possible (for an unforeseen technical reason), this phase was completed with these strains with the same conditions and concentration levels as the originally proposed procedure, therefore the confirmatory mutation test (pre-incubation test with E. coli WP2 uvrA) and the later performed confirmatory mutation test (pre-incubation test with S. typhimurium strains) phases were mentioned and evaluated as confirmatory mutation tests. .

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO).

At the concentration choice the solubility and the cytotoxicity of the test item (obtained in the concentration range finding test) were taken into consideration and based on the recommendations of OECD 471 guideline [6] the following concentrations of the test item were prepared and investigated in the initial mutation test:

in S. typhimurium strains: ±S9: 100, 75, 50, 16, 5, 1.6 and 0.5 μg/plate;

in E. coli WP2 uvrA: ±S9: 500, 160, 100, 50, 16, 5 and 1.6 μg/plate.

In the subsequent experiments the above strains were investigated with modified concentration ranges depending on the obtained actual cytotoxicity and mutagenicity results.

The confirmatory mutation tests (pre-incubation tests) were performed with following modified concentration levels:

in S. typhimurium strains: ±S9: 75, 50, 16, 5, 1.6, 0.5 and 0.16 μg/plate;

in E. coli WP2 uvrA: ±S9: 1000, 500, 160, 50, 16, 5 and 1.6 μg/plate.

Based on the noticed test item effects (or missing effects) subsequent additional partial experiments were performed: additional partial plate incorporation test with S. typhimurium TA1537 and E. coli WP2 uvrA, with following concentration levels:

in S. typhimurium TA1537: ±S9: 60, 50, 40, 30, 16, 10 and 5 μg/plate;

in E. coli WP2 uvrA: ±S9: 1000, 750, 500 and 160 μg/plate;

and additional repeated pre-incubation test with E. coli WP2 uvrA, with following concentration levels:

in E. coli WP2 uvrA: ±S9: 1000, 500, 160, 50, 16, 5 and 1.6 μg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In the performed experiments inhibitory, cytotoxic effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by decreased revertant colony counts (absent revertants or revertants below the corresponding historical control data ranges and/or corresponding vehicle) and/or affected colony development (pinpoint colonies) and/or affected background lawn development (reduced or slightly reduced background lawn). The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate (following the plate incorporation procedure in S. typhimurium TA1535 and TA1537; following the pre-incubation procedure in all S. typhimurium strains).

The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and all of the number of revertants fell in the corresponding historical control ranges (or even were above the historical control data range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

In the initial mutation test, following the plate incorporation procedure, positive results, significant, biologically relevant, dose-related changed revertant colony number increases, revertant colony numbers above the vehicle control data and above the historical control data ranges were obtained in S. typhimurium TA1537 strain in the absence and presence of exogenous metabolic activation (±S9). The biologically relevant increases were adequately confirmed in subsequent experiments. Furthermore, equivocal, borderline positive results, biological relevant revertant colony number increases were noticed in the confirmatory mutation test (pre-incubation test) in E. coli WP2 uvrA, in the absence of exogenous metabolic activation (-S9). These latter increases were not repeated, adequately confirmed, the higher revertant colony numbers remained below the relevant genotoxicological threshold for being positive in the subsequent experiment (repeated pre-incubation test).

In summary unequivocal biologically relevant increases in revertant colony numbers were observed in Salmonella typhimurium TA1537 strain following treatment with DIMETHOXYDIPHENYL-IODONIUMBROMID, in the absence and presence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induce gene mutations by frameshifts in the genome of the investigated Salmonella typhimurium TA1537 strain in the absence and presence of exogenous metabolic activation.

In conclusion, the test item DIMETHOXYDIPHENYL-IODONIUMBROMID has mutagenic activity on the applied Salmonella typhimurium TA1537 under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)