6-bromo-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

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:

6-bromo-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

EC Number:

201-381-8

EC Name:

6-bromo-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

Cas Number:

81-85-6

Molecular formula:

C17H10BrNO2

IUPAC Name:

6-bromo-3-methyl-3H-naphtho[1,2,3-de]quinoline-2,7-dione

Test material form:

solid

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

2; 6; 20; 66; 200; 666; 1000; and 2000 μg/plate (Pre-Experiment/Experiment I)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

- OTHER
- Study controls: The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.
- Concentrations of positive controls:
Sodium azide (NaN3) - 10µg/plate for TA 1535 and TA 100
4-Nitro-o-phenylene-diamine (4-NOPD) - 10 μg/plate for TA98, 50 μg/plate for TA1537
Methyl methane sulfonate (MMS) - 2 μg/plate for TA102
2-Aminoanthracene (2AA) - 10 μg/plate for TA102, 2.5 μg/plate for TA 98, TA 100, TA1535 and TA1537

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitation: The test item precipitated in the overlay agar in the test tubes from 666 to 2000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 2000 μg/plate. The undissolved particles had no influence on the data recording.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce reverse mutations in bacteria. During the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of the strains used (in strain TA1537). Therefore, pyridon is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 102 were treated with the test item using the Ames plate incorporation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The test item was tested at the following concentrations: 2, 6, 20, 66, 200, 666, 1000 and 2000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 666 to 2000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 2000 μg/plate. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 2000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with Pyridon in strain TA1537 in the presence and absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of the strains used, i.e. in strain TA1537. Therefore, Pyridon is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.