4,4'-isopropylidenebis[2-allylphenol]

Genetic toxicity: in vitro

Administrative data

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:

Experimental Starting date: 4 June 2014; Experimental Completion date: 24 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

4,4'-isopropylidenebis[2-allylphenol]

EC Number:

217-121-1

EC Name:

4,4'-isopropylidenebis[2-allylphenol]

Cas Number:

1745-89-7

Molecular formula:

C21H24O2

IUPAC Name:

4-{1-[4-hydroxy-3-(prop-2-en-1-yl)phenyl]propyl}-2-(prop-2-en-1-yl)phenol

Test material form:

liquid: viscous

Details on test material:

- Test Substance I.D.: 4,4’-isopropylidenebis[2-allylphenol] (TK 11907)
- Test Substance Batch No.: AG16A55043
- Test Substance Purity: 89% (provided by Sponsor)
- BioReliance Study No.: AD90LY.503REACH.BTL
- Test Substance Description: Clear colorless viscous liquid
- Storage Conditions: 2 to 8ºC, protected from light
- Test Substance Receipt/Login Date: 28 May 2014

Method

Target gene:

Reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA.
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

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9.

Test concentrations with justification for top dose:

- In the initial toxicity-mutation assay, the dose levels tested were: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.

- In the confirmatory mutagenicity assay, the dose levels tested were: 1.5, 5.0, 15, 50, 150, 500 and 1500 μg per plate with tester strains TA98, TA100 and TA1535 and 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.
The vehicle used to deliver TK 11907 to the test system was DMSO (CAS No. 67-68-5, Lot No. SHBD1324V, Purity: 99.98%, Exp. Date: May 2017), obtained from Sigma-Aldrich.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (μg/plate) for TA98 and TA1535; at 2 (μg/plate) for TA100 and TA1537 and at 15 (μg/plate) for WP2uvrA.

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1,000 (μg/plate) for WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (μg/plate) for TA 98

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 1 (μg/plate) for TA 100 and TA 1535

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Remarks:

At 75 (μg/plate) for TA 1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

into a vessel, containing 30 to 50 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 12 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 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Evaluation criteria:

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

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged equal to
2.0-times the mean vehicle control value.

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:

The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:
Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments), LIMS System (BioReliance), Excel 2007 (Microsoft Corporation), BRIQS (BioReliance) and Kaye Lab Watch Monitoring System (Kaye GE).

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In B2 (confirmatory mutagenicity assay) Precipitate was observed beginning at 500 or 1500 μg per plate with most test conditions. Toxicity was observed beginning at concentrations from 150 to 5000 μg per plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS AND DISCUSSION

Solubility Test

DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

Sterility Results

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

Tester starin titer results:

Experiment	Tester strain
Experiment	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Experiment	Titer Value (x 109 cells per mL)
B1	1.1	1.3	2.0	2.1	2.3
B2	2.3	1.1	8.7	2.9	4.0

Initial Toxicity-Mutation Assay

The results of the initial toxicity-mutation assay were generated in Experiment B1.

In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 μg per plate. Toxicity was observed beginning at concentrations from 50 to 5000 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 1500 μg per plate with tester strains TA98, TA100 and TA1535 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA.

Confirmatory Mutagenicity Assay

The results of the confirmatory mutagenicity assay are presented were generated in Experiment B2.

In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500 and 1500 μg per plate with tester strains TA98, TA100 and TA1535 and 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA. Precipitate was observed beginning at 500 or 1500 μg per plate with most test conditions. Toxicity was observed beginning at concentrations from 150 to 5000 μg per plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative With and without metabolic activation.

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, TK 11907 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:

The purpose of this study was to evaluate the mutagenic potential of the test substance 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.

The test substance, 4,4’-isopropylidenebis[2-allylphenol] (TK 11907), hereafter referred to as TK 11907, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, 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. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 μg per plate. Toxicity was observed beginning at concentrations from 50 to 5000 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 1500 μg per plate with tester strains TA98, TA100 and TA1535 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500 and 1500 μg per plate with tester strains TA98, TA100 and TA1535 and 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA. Precipitate was observed beginning at 500 or 1500 μg per plate with most test conditions. Toxicity was observed beginning at concentrations from 150 to 5000 μg per plate.

Under the conditions of this study, test substance TK 11907 was concluded to be negative in the Bacterial Reverse Mutation Assay.