((4S,4aR,7S,7aR)-4,7-dimethylhexahydrocyclopenta[c]pyran-1(3H)-one

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:

November 2020 to May 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

N/A

Molecular formula:

C10H16O2

Test material form:

liquid

Remarks:

Colorless

Details on test material:

Test substance was expected to be stable for the duration of testing.

Specific details on test material used for the study:

Batch Number - CH-DA-01-18

Method

Target gene:

Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli

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:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

The phenobarbitone/ beta-naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4272 and the protein level was adjusted to 20 mg/mL.

A 0.5 mL aliquot of the S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated , in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment 1 - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.

The dose range used for experiment 2 was determined by the results of experiment 1 and was as follows:

Salmonella strains: 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate
E.coli strains: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Eight test item concentrations were selected in experiment 2 in order to ensure the study achieved at least 4 non-toxic dose levels as required by the test guideline and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the solvent.

Controls

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

Details on test system and experimental conditions:

The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the solvent.

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in high purity DMSO by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for the stated water/impurity content (22.79%) of the test item. All test item preparation and dosing was performed under yellow safety lighting.

All formulations were used within 4 hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Experiment 1 - Plate Incorporation Method

DOSE SELECTION - Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

WITHOUT METABOLIC ACTIVATION - A 0.1 mL aliquot of the appropriate concentration of test item, solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of test item, appropriate positive and solvent controls and each bacterial strain, was assayed in triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

WITH METABOLIC ACTIVATION - As without metabolic activation with the exception that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

INCUBATION AND SCORING - All of the plates were incubated at 37 +/- 3 oC for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thining of the background material.

Experiment 2 - Pre-Incubation Method

DOSE SELECTION - The dose range used for experiment 2 was determined by the results of experiment 1 and was as follows:

Salmonella strains: 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate
E.coli strains: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

WITHOUT METABOLIC ACTIVATION - A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of appropriate concentration of test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 +/- 3 oC for 20 minutes (with shaking) prior to the addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating on Vogel-Bonner plates. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

WITH METABOLIC ACTIVATION - As without metabolic activation with the exception that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 +/- 3 oC for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

INCUBATION AND SCORING - All of the plates were incubated at 37 +/- 3 oC for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thining of the background material.

Rationale for test conditions:

In accordance with OECD Testing Guideline 471

Evaluation criteria:

There are several criteria for determining a positive result. Any, one or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979)

2. A reproducible increase at one or more concentrations.

3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TS1535 and TA1537.

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

The plate scoring system contains a built-in statistical analysis which is automatically performed during the scoring process using Dunnett's Regression Analysis. However, statistical analysis was not require in result assessment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed with test item exposure to all tester strains in the absence and presence of metabolic activation, initially from 150-500 ug/plate. All had weakened bacterial background lawns and/or a reduction in revertant counts.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed with test item exposure to all tester strains in the absence and presence of metabolic activation, initially from 150-500 ug/plate. All had weakened bacterial background lawns and/or a reduction in revertant counts.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed with test item exposure to all tester strains in the absence and presence of metabolic activation, initially from 150-500 ug/plate. All had weakened bacterial background lawns and/or a reduction in revertant counts.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed with test item exposure to all tester strains in the absence and presence of metabolic activation, initially from 150-500 ug/plate. All had weakened bacterial background lawns and/or a reduction in revertant counts.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed with test item exposure to all tester strains in the absence and presence of metabolic activation, initially from 150-500 ug/plate. All had weakened bacterial background lawns and/or a reduction in revertant counts.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay 'Ames Test' using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item dihydronepetalactone did not induce an increase in frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of the test, dihydronepetalactone was considered to be non-mutagenic.

Executive summary:

The potential of dihydronepetalactone to induce gene mutation in bacteria was assessed using a GLP-compliant study performed in accordance with OECD Testing Guideline 471.

Experiment 1 (plate incorporation)

The maximum dose level of the test item in the first experiment was selected as the OECD 471 recommended dose level of 5000 ug/plate as recommended for a soluble and non-toxic substance.

Toxicity, evaluated as a visible reduction in growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all of the salmonella testing strains with and without metabolic activation initially from 1500 ug/plate.  Toxicity was noted to WP2uvrA both without and without metabolic activation at 5000 ug/plate.

No test item precipitate was observed on any of the plates at any of the doses tested in eith the presence or absence of metabolic activation.

There were no biologically relevant increases in the frequency of reverant colonies recorded for any of the bacterial strains, with any dose of test item, either with or without metabolic activation.

 

Experiment 2 (pre-incubation)

The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 ug/plate) for WP2uvrA but 1500 ug/plate for all of the Salmonella tester strains.

A stronger toxic response was noted in the second mutation test after employing the pre-incubation modification with weakened bacterial background lawns and/or reduction in revertant counts, observed with test item exposure to all tester strains without or without metabolic activation, initially from 150-500 ug/plate, respectivaly.

No test item precipitate was observed on any of the plates at any of the doses tested in eith the presence or absence of metabolic activation.

There were no biologically relevant increases in the frequency of reverant colonies recorded for any of the bacterial strains, with any dose of test item, either with or without metabolic activation.

 

The test item dihydronepetalactone did not induce an increase in frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of the test, dihydronepetalactone was considered to be non-mutagenic.