Ethyltriphenylphosphonium acetate

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:

21 August 2017 to 20 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylphosphonium acetate

EC Number:

252-743-7

EC Name:

Ethyltriphenylphosphonium acetate

Cas Number:

35835-94-0

Molecular formula:

C20H20P.C2H3O2

IUPAC Name:

ethyltriphenylphosphanium acetate

Test material form:

solid

Method

Target gene:

his

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. The top dose was the maximum dose per the guidelines.
definitive mutagenicity assay: 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (for the test substance)
All positive controls were diluted in DMSO, except for sodium azide, which was diluted in sterile water.
- Justification for choice of solvent/vehicle: Ethanol was the vehicle of choice based on the Sponsor’s request and the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in ethanol at a concentration of approximately 100 mg/mL in the solubility test conducted at BioReliance.

Controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracebe

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary-toxicity assay; 3 in the mutagenicity assay

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn , revertant colony numbers

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 consecutive 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 uvrA
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 an 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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed

RANGE-FINDING/SCREENING STUDIES:
a 50.0 μL plating aliquot. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium; no toxicity was observed for E. coli WP2.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attachment
- Negative (solvent/vehicle) historical control data: see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium. Cytotoxicity was observed for E. coli WP2 only at 5000 µg/plate without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, ETPPAAc 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, ETPPAAc, 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 in accordance with OECD guideline 471. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with most 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 ETPPAAc 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.