Rosetal A

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:

08 May 2017 to 29 May 2017

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

[2,2-bis[(3,7-dimethyl-2,6-octadienyl)oxy]ethyl]benzene

EC Number:

266-805-6

EC Name:

[2,2-bis[(3,7-dimethyl-2,6-octadienyl)oxy]ethyl]benzene

Cas Number:

67634-02-0

Molecular formula:

C28H42O2

IUPAC Name:

(2,2‐bis(((E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl)oxy)ethyl)benzene

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3779, Exp. Date: 06 Apr 2019) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared on the day of use. Final concentrations of the components were β-nicotinamide-adenine dinucleotide phosphate 4mM, Glucose-6-phosphate 5 mM, Potassium chloride 33mM, Magnesium chloride 8mM, Phosphate Buffer (pH 7.4) 100 mM and S9 homogenate 10% (v/v).

Test concentrations with justification for top dose:

The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvr A were exposed to the vehicle alone, positive controls and eight dose levels of the test substance (1.5, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate), in duplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon the results of the initial toxicity-mutation assay and lack of post-treatment toxicity. Dose levels selected were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. In both assays no toxicity was observed. Precipitate was observed in both assays beginning at 1500 or at 5000 μg per plate with all conditions.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of solvent/vehicle: Ethanol was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells.
- Justification for percentage of solvent in the final culture medium: The test substance formed a clear solution in ethanol at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

see details on plating in Table 1 in any other information

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
The test system was exposed to the test substabce via the plate incorporation methodology. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate in initial toxicity-mutation assay and triplicate in confirmatory mutagenicity assay
- Number of independent experiments: 2 (initial toxicity-mutation assay and confirmatory mutagenicity assay)

SCORING:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.

TEST STRAIN VERIFICATION
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

Strains TA1535 and TA1537:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvr A:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Initial Toxicity-Mutation Assay: Precipitate was observed beginning at 1500 or at 5000 µg per plate with all conditions but did not interfere with the scoring.
- Confirmatory Mutation Assay: Precipitate was observed beginning at 1500 or at 5000 µg per plate with all conditions but did not interfere with the scoring.

Ames test:
- Signs of toxicity: Non observed
- Individual plate counts: Yes, described
- Mean number of revertant colonies per plate and standard deviation: Yes, described

HISTORICAL CONTROL DATA): Yes, described
- Positive historical control data: All tester strain cultures were within ranges of historical control values (2015).
- Negative (solvent/vehicle) historical control data: All tester strain cultures were within ranges of historical control values (2015).

Any other information on results incl. tables

The results presented are true for both the Initial Toxicity-Mutation assay and the Confirmatory Mutation Assay.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay performed according to OECD TG 471 (1997).

Executive summary:

The test substance, Rosetal A, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Ethanol was used as the vehicle.

Results: In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 or at 5000 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 or at 5000 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. These results indicate that the substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.