ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

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 October 2003 to 04 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

EC Number:

806-801-9

Cas Number:

1539267-56-5

Molecular formula:

C25H25O4P

IUPAC Name:

ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off white powder.
- Storage conditions: Room temperature, in the dark.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability of the test material was not determined as part of this study.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability and homogeneity of the test material in the vehicle was not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Method

Target gene:

Histidine locus (Salmonella typhimurium strains)
Tryptophan locus (Escherichia coli strain)

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 fraction, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6-benzoflavone (80 mg/kg 2 days before killing) to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80 °C.
- method of preparation of S9 mix: The S9 mix contained: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.

Test concentrations with justification for top dose:

Test 1 (plate incorporation assay): 5, 15, 50, 150, 500, 1 500, 5 000 µg/ plate (with and without metabolic activation)
Test 2 (pre-incubation assay): 50, 150, 500, 1 500, 5 000 µg/ plate (with and without metabolic activation)

The highest concentration of test material tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5 000 µg/ plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The Sponsor indicated that the test material was insoluble in water but soluble in dimethyl sulphoxide (DMSO). DMSO (ACS reagent grade) was, therefore, used as the vehicle for this study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

monomeric acrylamide

benzo(a)pyrene

other: 2-Aminoanthracene; solvent: DMSO (with metabolic activation; 5 µg/plate for strains TA100 and TA1535; 10 µg/plate for strain WP2 uvrA (pKM101))

Details on test system and experimental conditions:

TEST 1 (PLATE INCORPORATION ASSAY)
Aliquots of 0.1 mL of the test material solutions, positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test material, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 °C for ca. 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

TEST 2 (PRE-INCUBATION ASSAY)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5 000 µg/plate, but only five concentrations were used.

Evaluation criteria:

ACCEPTANCE CRITERIA
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

CRITERIA FOR ASSESSING MUTAGENIC POTENTIAL
If exposure to a test material produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test material does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups.
The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test material formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

TEST 1 (PLATE INCORPORATION ASSAY)
No evidence of toxicity was obtained following exposure to the test material. No precipitate was observed on any plates. A maximum exposure concentration of 5 000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 µg/plate in either the presence or absence of S9 mix.

TEST 2 (PRE-INCUBATION ASSAY)
No evidence of toxicity was obtained following exposure to the test material. No precipitate was observed on any plates containing the test material at 5 000 µg/plate.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 µg/plate in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

It was concluded that the test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100, under GLP conditions.

During the study, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material, which was diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of test material up to 5 000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to the test material. No precipitate was observed on any plates containing the test material.

No evidence of mutagenic activity was seen at any concentration of test material in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was therefore concluded that the test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.