Tris(2-methylphenyl)phosphine

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:

11 Sep 2017 to 15 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:

no

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-methylphenyl)phosphine

EC Number:

228-193-9

EC Name:

Tris(2-methylphenyl)phosphine

Cas Number:

6163-58-2

Molecular formula:

C21H21P

IUPAC Name:

tris(2-methylphenyl)phosphane

Test material form:

solid: particulate/powder

Details on test material:

Lot number: 2000850599
Analysis date: 19 August 2016

Specific details on test material used for the study:

Identification: Tri-o-tolylphosphine
Appearance: Off-white crystalline powder (determined by Charles River Den Bosch)
Batch: B64002 (Pfizer Lot no. 2000850599)
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature
Stable under storage conditions until: Unknown
Test Facility test item number: 208378/A

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:

E. coli strain WP 2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

exogenous mammalian metabolic activation system (S9)

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
First Experiment: Direct Plate Assay- of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Absence of S9-mix: 5.4, 17, 52, 164 and 512 µg/plate.
Presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate.
Second Experiment: Pre-Incubation Assay: Tested up to the dose levels of 512 and 1600 µg/plate in the absence and presence of S9-mix

Vehicle / solvent:

dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

Test System Salmonella typhimurium bacteria and Escherichia coli bacteria
Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 h
- Expression time (cells in growth medium): Preparation of bacterial cultures: 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 E9 cells/ml) Freshly grown cultures of each strain were used for a test.

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

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

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment. A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Statistical analysis results that are generated by the program but are not required by study plan and/or are not scientifically relevant will be retained on file but will not be included in the tabulations.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.

HISTORICAL CONTROL DATA
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for WP2uvrA in the absence of S9-mix in the second experiment.
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that Tri-o-tolylphosphine 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 Tri-o-tolylphosphine 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 B64002 (Pfizer Lot no. 2000850599) of Tri-o-tolylphosphine was an off-white crystalline powder. The vehicle of the test item was dimethyl sulfoxide. 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 was tested up to or beyond a precipitating 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 in the strains TA1535, TA1537 and TA98 up to concentrations of 512 and 5000 µg/plate in the absence and presence of S9-mix, respectively. The test item precipitated on the plates at dose levels of 164 and 1600 µg/plate and upwards in the absence and presence of S9-mix, respectively. 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 in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay up to concentrations of 512 and 1600 µg/plate in the absence and presence of S9-mix, respectively. The test item precipitated on the plates at dose levels of 512 µg/plate and upwards. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all Salmonella typhimurium tester strains in the absence 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 Tri-o-tolylphosphine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli  reverse mutation assay.