Dibenzylbenzene, ar-methyl derivative

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:

15 March 2019 - 09 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st july 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Dibenzylbenzene, ar-methyl derivative

EC Number:

258-649-2

EC Name:

Dibenzylbenzene, ar-methyl derivative

Cas Number:

53585-53-8

Molecular formula:

C21H20

IUPAC Name:

dibenzylbenzene, ar-methyl derivative

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The selected dose levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at required concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Merhod: observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Rationale for test conditions:

Not applicable.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

None.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Emulsion

RANGE-FINDING STUDY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 500 µg/plate. This emulsion prevented the scoring of the revertants at 5000 µg/plate in the TA 98 strain.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the three strains used, either with or without S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 250 µg/plate.
No noteworthy toxicity was noted at any dose levels, towards the five strains used.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14).  A preliminary toxicity test was performed to define the dose levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, at 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results

The test item was found non-toxic in the preliminary test, but poorly soluble in the final treatment medium (emulsion in the Petri plates). Consequently, the selection of the highest dose level to be used in the main experiments was based on the level of emulsion, according to the criteria specified in the international guidelines.

 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analyzable dose levels for each strain and test condition. The study was therefore considered to be valid.

 

The selected dose levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

 

A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 250 µg/plate.

 

No noteworthy toxicity was noted at any dose levels, towards the five strains used.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment, with or without S9 mix. These results met the criteria of a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or absence of a rat liver metabolizing system.