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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of Dibenzylbenzene, ar-methyl derivative, hydrogenated was assessed in an Ames test according to OECD guideline and in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods. The test item was tested at up to eight dose levels, with 5000 µg/plate being the highest dose level, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). A test item precipitate (globular in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies. In both experiments (plate incorporation and pre-incubation method) were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). Dibenzylbenzene, ar-methyl derivative, hydrogenated was considered to be non-mutagenic under the conditions of this test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August to September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate Good Laboratory Practice: The Department of Health of the Government ot the United Kingdom, Statement of compliance in accordance with Directive 2004/9/EC, date of inspection 05/07/2016
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.of test material: hydrogenius 005
- Expiration date of the lot/batch: > 3 years (unlimited shelf life)
- Storage condition of test material: room temperature in the dark
- Purity: 100%
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: TA1537 (his C 3076; rfa-; uvrB-) and TA98 (his D 3052; rfa-; uvrB-; R-factor): frame shift mutation TA1535 (his G 46; rfa-, uvrB-), TA100 (his G 46; rfa-; uvrB-; R-factor) and E.coli WP2uvrA (trp-; uvrA-): base pair substitution
Remarks:
bacteria were obtained from University of California, Berkley, on 04 August 1995, resp. British Industrial Biological Research Association, on 17 august 1987, all strains stored at approx. -196°C in a liquid nitrogene freezer
Metabolic activation:
with and without
Metabolic activation system:
S9 male rat liver microsomal fraction; animals induced with phenobarbitone/b-naphthoflavone, batch: PB/BNF 05/06/2016, Envigo Research Ltd. Certificate of S9 Efficacy
Test concentrations with justification for top dose:
Exp. 1 (plate incorporation method)I: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Exp. 2 (pre-incubation method): 15, 50, 150, 500, 1500 and 5000 µg/plate
The top dose is the recommended maximum test concentration for non-cytotoxic substances according to the guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: based on solubility testing. The test material was immiscible in sterile distilled water, dimethyl sulphoxid and dimethyl formamide at 50 mg/mL, but miscible in acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test item precipated out of solution after formulation in acetone when stored at room temperature, therefore THF was selected as vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
All positive control substances were dissolved in dimethyl sulfoxide (DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: exp.1 in agar (plate incorporation), exp.2 pre-incubation method; according to the OECD Guideline

DURATION
- Preincubation period (only exp. 2): 20 min at 37 ± 3°C with shaking
- Exposure duration: 48 h at 37 ± 3°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn, the plates were viewed microscopically for evidence of thinning

ACCEPTABILITY CRITERIA
- All bacterial strains must demonstrate the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All of the positive control substances should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:
- a dose-related increase in mutant frequency over the dose range tested
- a reproducible increase at one or more concentrations.
- biological relevance against in-house historical control ranges.
- statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.


Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
other: all strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar, the S9-mix used in both experiments and the test item formulations were shown to be sterile.
Precipitation (globular in appearance) was noted at the highest test item concentration (5000 ug/plate), this observation did not prevent the scoring of revertant colonies
Positive and negative control results were considered valid.
Cytotoxicity: no visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix).
Positive historical control data: results were within the historical control ranges (data from years 2014 and 2015, see attachment "Historical control data") and therefore considered acceptable.
Negative (solvent/vehicle) historical control data: results were within the historical control ranges (data from 2014 and 2015, see attachment "Historical control data") and therefore considered acceptable.

Detailed results can be seen in the attached document "Test results".

Conclusions:
Dibenzylbenzene, ar-methyl derivative, hydrogenated was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to 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 dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation, thus validating the sensitivity of the assay and the efficacy of the S9-mix. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). A test item precipitate (globular in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies. In both experiments (plate incorporation and pre-incubation method) were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Therefore, dibenzylbenzene, ar-methyl derivative, hydrogenated was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the available information on genetic toxicity, Dibenzylbenzene, ar-methyl derivative, hydrogenated does not present a genetic toxicity hazard according Regulation (EC) No 1272/2008 and does not require classification.