Tris(methylphenyl) phosphite

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:

January 21, 2008 through February 19, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(methylphenyl) phosphite

EC Number:

247-119-6

EC Name:

Tris(methylphenyl) phosphite

Cas Number:

25586-42-9

Molecular formula:

C21H21O3P

IUPAC Name:

tris(methylphenyl) phosphite

Details on test material:

- Name of test material (as cited in study report): Tritolyl Phosphite (BU)
- Physical state: liquid; amber
- Analytical purity: 91.8%
- Lot/batch No.: 11237993-7079
- Expiration date of the lot/batch: November 23, 2008
- Stability under test conditions: not indicated by sponsor
- Storage condition of test material: at room temperature, moisture protected

Method

Target gene:

TA 1537 - his C 3076; rfa-; uvrB-: (frame shift mutations)
TA 98 - his D 3052; rfa-; uvrB-;R-factor (frame shift mutation)
TA 1535 - his G 46; rfa-; uvrB-: (base-pair substitutions)
TA 100 - his G 46; rfa-; uvrB-;R-factor (base-pair substitutions)
WP2 uvrA - trp-; uvrA-: (base-pair substitutions and others)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500, and 5000 ug/plate

Vehicle / solvent:

Test item was disolved in dry THF (MERCK, D-64293 Darmstadt; purity > 99%). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for strains TA1535 and TA100 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

for strains TA1537 and TA98 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for strain E. coli WP2 uvr A without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains with metabolic activation

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates: 25 uL Test solution at each dose level, solvent (negative control) 100 uL or reference mutagen solution (positive control), 500 uL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 uL Bacteria suspension (cf. test system, pre-culture of the strains), 2000 uL Overlay agar. In the pre-incubation assay 25 pL test solution, solvent or 100 uL reference mutagen solution, 500 uL S9 mix / S9 mix substitution buffer and 100 uL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

During the described mutangenicty test and under the experimental conditions reported. the test item did not induce gene mutations by base pair changes or frameshift in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of Tritolyl Phospite (BU) to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations of the active ingredient: Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate; Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tritolyl Phospite (BU) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.