2,3-dihydro-2,2,6-trimethylbenzaldehyde

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:

From December 28, 2018 to February 22, 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

EC Number:

204-133-7

EC Name:

2,3-dihydro-2,2,6-trimethylbenzaldehyde

Cas Number:

116-26-7

Molecular formula:

C10H14O

IUPAC Name:

2,6,6-trimethylcyclohexa-1,3-diene-1-carbaldehyde

Test material form:

liquid

Method

Target gene:

The tester strains used for the study were Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537 and TA102.

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 S9-mix induced by induced by Aroclor 1254

Test concentrations with justification for top dose:

In the preliminary cytotoxicity assay, no precipitation was observed at the test item highest concentration of 5000 µg/plate. Complete inhibition of background lawn (cytotoxicity) and reduction in the number of revertant colonies were observed at the concentration of 5000, 2500, 1250, 625.0 µg/plate. Similarly, partial inhibition of background lawn was observed at 312.5 µg/plate when compared to vehicle control. Based on the results of preliminary cytotoxicity assay, test item concentrations of 19.53, 39.06, 78.13, 156.3, and 312.5 µg/plate are selected for testing in the main mutagenicity study.

Vehicle / solvent:

Dimethyl sulphoxide (Lot No.SHBJ6335, Sigma-Aldrich) was used as vehicle to prepare different dilutions of the test item. Vehicle control was plated for all strains in the presence and absence of S9 mix.

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

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 were procured from Molecular Toxicology, Inc. PO Box 1189 Boone, NC 28607 USA.
Stock cultures of tester strains in oxoid nutrient broth no. 2 with 10% DMSO are stored in the test facility as frozen permanents at -80±10ºC.

Rationale for test conditions:

The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many test items including a wide range of chemical classes. Additionally, the tester strains of Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 are the recommended by regulatory agencies for conducting Bacterial Reverse Mutation Test.

Evaluation criteria:

The following criteria were used to determine a valid assay before the data evaluation for interpretation of results.
- Regular background growth in the solvent control and negative control (if any).
- To demonstrate that appropriate numbers of bacteria are plated, density of the tester strain cultures must be greater than or equal to 0.5 x 109 bacteria/mL.
- The positive control substances should produce a significant increase in mutant colony frequencies over the vehicle control plates.
- The spontaneous reversion rates in the solvent control and negative control (if any) is in the range of our historical data.
- Minimum five analyzable test item doses should be available to evaluate mutation frequencies.

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:

S. typhimurium TA 102

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:

The test item, Safranal was assessed for its potential to induce gene mutation by plate incorporation and pre-incubation method using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 both in the presence and absence of metabolic activation along with vehicle and positive controls. No significant increase in the revertant colony count was observed in any of the tested concentrations, both in the presence and absence of metabolic activation, when compared to the vehicle control.
The spontaneous revertant colony count of vehicle and positive controls was within the range of laboratory historical control data.

Applicant's summary and conclusion

Conclusions:

It is thus concluded that the test item, Safranal is Non-Mutagenic up to the highest tested concentrations of 312.5 µg/plate in the five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence (10% S9 mix) and absence of metabolic activation, as measured by the Bacterial Reverse Mutation Test, under the conditions of the test employed.

Executive summary:

The test item, Safranal from Organica Aromatics Private Limited, was evaluated in a Bacterial Reverse Mutation Assay as per OECD Guideline No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item, Safranal was tested for its ability to induce reverse mutations at the histidine locus in tester strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) in the presence and absence of an exogenous mammalian metabolic activation system (S9).

The test item was found to be soluble in DMSO at a concentration of 50 mg/mL and no precipitation was observed at the highest tested concentration i.e. 5000 µg/plate. On the basis of solubility and precipitation tests, the Preliminary Cytotoxicity Test was performed at 39.06, 78.13, 156.3, 312.5, 625.0, 1250, 2500 and 5000µg/plate with Salmonella typhimurium TA100 both in the presence and absence of a metabolic activation system.

Complete inhibition of background lawn and reduction in the number of revertant colonies were observed at the concentration of 5000, 2500, 1250, 625.0 µg/plate. Similarly, partial inhibition of background lawn was observed at 312.5 µg/plate when compared to vehicle control. Therefore, on the basis of preliminary cytotoxicity assay results, the Trial I and Trial II were performed at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate both in the presence and absence of metabolic activation. Vehicle control (dimethyl sulfoxide) and appropriate positive controls (2- nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2- Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

In Trial I, tester strains TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate, along with vehicle and positive controls both in the presence and absence of metabolic activation by plate incorporation method. No significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

In Trial II, all the tester strains, TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate by pre-incubation method along with vehicle and positive controls, both in the presence and absence of metabolic activation. In all the tester strains, no significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

The spontaneous revertant frequency of the vehicle control group was within range of historical control data. The positive controls used in the study exhibited significant increase in mean number of revertant frequency respective to their strains, indicating that the sensitivity of the tester strains towards specific mutagens and confirmed that the test conditions were adequate and that the metabolic activation system functioned properly.

It is thus concluded that the test item, Safranal is Non-Mutagenic up to the highest tested concentrations of 312.5 µg/plate in the five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence (10% S9 mix) and absence of metabolic activation, as measured by the Bacterial Reverse Mutation Test, under the conditions of the test employed.