Phenylphosphonic acid

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:

18 Sep 2017 to 29 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

There was a deviation in the mean plate count of the positive control but this deviation had no effect on the results of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

yes

Remarks:

There was a deviation in the mean plate count of the positive control but this deviation had no effect on the results of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenylphosphonic acid

EC Number:

216-388-1

EC Name:

Phenylphosphonic acid

Cas Number:

1571-33-1

Molecular formula:

C6H7O3P

IUPAC Name:

phenylphosphonic acid

Test material form:

solid: particulate/powder

Details on test material:

Lot number LKF6065

Specific details on test material used for the study:

Identification: Phenyl Phosphonic Acid
Appearance: White crystalline powder
Batch: LKF6065
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 09 December 2017 (expiry date)

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

Remarks:

Escherichia coli (E. coli) strain WP 2 uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Exogenous mammalian metabolic activation system (S9).

Test concentrations with justification for top dose:

5000 µg/plate

Justification: In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Vehicle / solvent:

The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).

Controls

Untreated negative controls:

yes

Remarks:

Milli-Q water

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: ICR-191, 2-nitrofluorene, 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 h
- Expression time (cells in growth medium): Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10 9 cells/ml). Freshly grown cultures of each strain were used for a test.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity- measured as a reduction of the bacterial background lawn.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Statistics:

Not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA1535, TA1537 and TA98

Remarks:

1st mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA1535, TA98 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

other: TA1537 and TA100

Remarks:

second mutation experiment

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as evidenced by a reduction of the bacterial background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that Phenyl Phosphonic Acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Phenyl Phosphonic Acid and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).  

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch LKF6065 of Phenyl Phosphonic Acid was a white crystalline powder.  The test item was dissolved in Milli-Q water.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay.  The test item did not precipitate on the plates at this dose level.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.  

In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98.  The test item did not precipitate on the plates at this dose level.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.  

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the

pre-incubation assay.  The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was only

observed in the tester strains TA1537 and TA100 in the presence of S9-mix.  

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.  These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Phenyl Phosphonic Acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the

Escherichia coli reverse mutation assay.