4,10-dibromodibenzo[def,mno]chrysene-6,12-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or no or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,10-dibromodibenzo[def,mno]chrysene-6,12-dione

EC Number:

224-481-3

EC Name:

4,10-dibromodibenzo[def,mno]chrysene-6,12-dione

Cas Number:

4378-61-4

Molecular formula:

C22H8Br2O2

IUPAC Name:

4,10-dibromonaphtho[7,8,1,2,3-nopqr]tetraphene-6,12-dione

Test material form:

solid: nanoform

Method

Target gene:

his- for S. typhimurium strains TA 1537, TA 1535, TA 100, TA 98
trp- for E. coli WP2 uvr A

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone-induced rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity >99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to 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:

other: Without metabolic activation: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate / With metabolic activation: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: yes

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 WP2 uvrA) 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:

Not mandatory according to OECD guideline 471

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments except for strains TA 1535, TA 98 and TA 100, where reduced background growth was observed at 5000 μg/plate in the presence of metabolic activation in experiment II

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase was observed in strain TA 98 with metabolic activation in experiment II at 1000 µg/plate. Since this single increase is caused by only one plate, this effect is judged as biologically irrelevant.

The historical control range was exceeded in strains TA 1535 (negative and solvent control, experiment I, and negative control, experiment II). TA 100 (negative control, experiment II) without S9 mix and in strains TA 1537 (negative control, experiment I), TA 100 (negative control, experiment II) and WP2 uvrA (negative control, experiment I) with S9 mix. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative both with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Pigment Red 168 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Pigment Red 168 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed according to OECD TG 471 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments except for strains TA 1535, TA 98, and TA 100, where reduced background growth was observed at 5000 μg/plate in the presence of metabolic activation in experiment II. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment Red 168 at any dose level, neither in the presence nor 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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.