Phosphine, tris(5-chloro-2-methoxyphenyl)-

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:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guidelines No. 471 "Bacterial Reverse Mutation Test"” and in accordance with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Guideline followed.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report)
- Physical state: White powder
- Analytical purity: 99.5%
- Storage condition of test material: Room temperature in the dark.

Method

Target gene:

The purpose of the study was to evaluate the test item, for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

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:

S9 metabolic system

Test concentrations with justification for top dose:

Preliminary Toxicity Test: The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 1-Jg/plate.

Mutation Test- Experiment:
Five concentrations of the test item (50, 150, 500, 1500 and 5000 1-Jg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml and in acetone at 100 mg/ml but was fully miscible in dimethyl formamide at 50 mg/ml in solubility checks performed in-house. Dimethyl formamide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Preparation of Test and Reference Items:
The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml and in acetone at 100 mg/ml but was fully miscible in dimethyl formamide at 50 mg/ml in solubility checks performed in-house. Dimethyl formamide was therefore selected as the vehicle.


The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 5 minutes at 40°C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.3%) of the test item. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron D M et a/ (1981)). All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 X 10-4 microns.

Microsomal Enzyme Fraction:
The S9 Microsomal fraction was prepared in-house (PB/ NF Lot 26 June 2011) from rats induced with Phenobarbitone/ -Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCI solution (1g liver to 3 ml KCI) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the supernatant were frozen and stored at approximately -196°C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.


59-Mix and Agar:
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

Test Procedure:

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 1-Jg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mllplate). Ten concentrations of the test item formulation and a vehicle control (dimethyl formamide) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 3rC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 1-Jg/plate because of excessive test item precipitation.


Mutation Test- Experiment 1
Five concentrations of the test item (50, 150, 500, 1500 and 5000 1-Jg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.


Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test item formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.


All of the plates were incubated at 3rC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 1-Jg/plate because of excessive test item precipitation.



Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 j..lg/plate).

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test item formulation and incubated for 20 minutes at 3rC with shaking at approximately
130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan
supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method.

All of the plates were incubated at 3rC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at and above 1500 j..lg/plate because of excessive test item precipitation.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De
Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon eta/ (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon eta/ (1989)).

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The test item, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method 813/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, tris(5-chloro-2- methoxyphenyl)phosphine, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 g/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 g/plate. A test item precipitate (particulate in appearance) was noted from 1500 and 500 g/plate in Experiments 1 and 2 respectively. This observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion: The test item, tris(5-chloro-2-methoxyphenyl)phosphine, was considered to be non-mutagenic under the conditions of this test.