2,4-dimethyl-4-phenyltetrahydrofuran

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 18 September 2020 to 02 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test guideline No. 471 without any deviation.

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)

Version / remarks:

updated and adopted 21 July 1997

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

rel-(2R,4R)-2,4-dimethyl-4-phenyltetrahydrofuran

Cas Number:

99343-90-5

Molecular formula:

C12H16O

IUPAC Name:

rel-(2R,4R)-2,4-dimethyl-4-phenyltetrahydrofuran

Constituent 2

Reference substance name:

rel-(2S,4R)-2,4-dimethyl-4-phenyltetrahydrofuran

Cas Number:

99343-91-6

Molecular formula:

C12H16O

IUPAC Name:

rel-(2S,4R)-2,4-dimethyl-4-phenyltetrahydrofuran

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
-Aspect Colorless to pale yellow liquid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) S9: S9-mix from the livers of male Sprague-Dawley rats induced with a single injection intraperitoneal of Aroclor 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
The S9 (Lot No. 4287, Exp. Date: 11 Aug 2022) 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 2aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use as indicated below:

- beta-nicotinamideadenine dinucleotide phosphate (4 mM)
- Glucose-6-phosphate (5 mM)
- Potassium chloride (33 mM)
- Magnesium chloride (8 mM)
- Phosphate Buffer at pH 7.4 (100 mM)
- S9 homogenate (10% v/v)

Test concentrations with justification for top dose:

- initial toxicity-mutation assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO in TA1535, TA1537, TA98, TA100 and WP2 uvrA, with and without S9-mix via the plate incorporation method.
- confirmatory mutagenicity assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO in TA1535, TA1537, TA98, TA100 and WP2 uvrA, with and without S9-mix via the plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO), Lot number SHBL2820 supplied by Sigma-Aldrich.
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance in DMSO formed a clear solution at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 2.0 µg/plate for TA98 and TA1535, 5.0 µg/plate for TA100 and TA1537 and 15 µg/plate for WP2 uvrA

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

PREPARATION OF TESTER STRAIN: On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 45 to 65  mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175  rpm and incubating at 37±2°C for approximately 10 +/-2.5 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

STERILITY CHECKS: To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.

METHOD OF APPLICATION: in agar (plate incorporation)
Media used in the treatment of the test system were indicated in the table 7.6/01 below.

MUTAGENICITY ASSAY: Onehalf (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.

DURATION
- Exposure duration: 48 to 72 h at 37 ± 2 °C

NUMBER OF REPLICATIONS:
- initial toxicity-mutation assay: 2 plates/dose
- Mutagenicity assay: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test material 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. See Table 7.6/02.

Rationale for test conditions:

The initial toxicity-mutation assay was used to establish the doserange for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation.
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. 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:

- For TA1535 and TA1537 strains, 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.

- For TA98, TA100 and WP2 uvrA strains, 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.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: Not observed.
- Other confounding effects: None

INITIAL TOXICITY-MUTATION ASSAY: No precipitate was observed, but toxicity was generally observed at 5000 µg/plate.
Several increases were observed with WP2 uvrA in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates appear to have greater fold increase.  There was no dose dependent trend.  These increases were not considered to be biologically significant.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

CONFIRMATORY MUTAGENICITY ASSAY: No precipitate was observed, but toxicity was observed from 1500 µg/plate for TA98 and from 5000 µg/plate for TA100 and TA 1535 in the presence of metabolic activation and from 1500 µg/plate for TA100 and WP2 uvrA and from 5000 µg/plate for TA98, TA1535 and TA1537 in the absence of metabolic activation.
Several increases were observed with TA1537 in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates appear to have greater fold increase.  There was no dose dependent trend.  These increases were not considered to be biologically significant.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical negative and positive control values of 2018.

See table of results in the full study report attached.

Any other information on results incl. tables

All criteria for a valid study were met as described in the protocol.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Rhubafuran did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvrA were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, 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 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance (1.50 to 5000 µg/plate), in duplicate, in the presence and absence of Aroclo-rinduced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.  TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance (15.0 to 5000 µg/plate), in triplicate, in the presence and absence of Aroclor-induced rat liver S9.  Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity assay.

 

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 precipitate was observed. Toxicity was observed at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with WP2 uvrA in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. 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 precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with TA1537 in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. 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 Rhubafuran 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.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.