Phenyl bis(2,4,6-trimethylbenzoyl)-phosphine oxide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1987)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenyl bis(2,4,6-trimethylbenzoyl)-phosphine oxide

EC Number:

423-340-5

EC Name:

Phenyl bis(2,4,6-trimethylbenzoyl)-phosphine oxide

Cas Number:

162881-26-7

Molecular formula:

C26 H27 O3 P

IUPAC Name:

[phenyl(2,4,6-trimethylbenzoyl)phosphoryl](2,4,6-trimethylphenyl)methanone

Details on test material:

- Name of test material (as cited in study report): TKA 40135 (CGI 819)

Method

Target gene:

histidine or tryptophan auxotrophy

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: defect in the excision repair system (uvrB) , presence of rfa character

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: defect in the excision repair system (uvrB) , presence of rfa character, AT base pair at the primary reversion site

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: AT base pair at the primary reversion site , DNA repair-proficient

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction obtained from the liver of male sprague-dawley rats treated with AROCLOR 1254, mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)

Test concentrations with justification for top dose:

Range-finding test: 20.6 - 5000 µg/plate
Main test: 312.5 - 5000 µg/plate
Confirmation: 312.5 - 5000 µg/plate

Vehicle / solvent:

DMSO was used as vehicle since the test substance was soluble up to the concentration of 50 mg/mL in this vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

see solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Controls 2

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

in DMSO at 1.5 µg/plate in TA 100, TA 1537, TA 98; in DMSO at 5.0 µg/plate in TA 102; in DMSO at 20 µg/plate in E. coli

Controls 3

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

cyclophosphamide

Remarks:

in bidistilled water at 200 µg/plate in TA 1535

Controls 4

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

sodium azide

Remarks:

in bidistilled water at 2 µg/plate in TA 100 and TA 1535

Controls 5

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: 4-Nitroquinoline

Remarks:

in DMSO at 2 µg/plate in E. coli

Controls 6

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

mitomycin C

Remarks:

in bidistilled water at 0.5 µg/plate in TA 102

Controls 7

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

2-nitrofluorene

Remarks:

in DMSO at 5 µg/plate in TA 98

Controls 8

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

9-aminoacridine

Remarks:

in DMSO at 80 µg/plate in TA 1537

Details on test system and experimental conditions:

PREPARATION OF THE TEST SOLUTIONS
The test material was dissolved in the dark in DMSO after slightly warmed up. The test substance was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. The test
substance and the solutions were handled in darkness unter red light. No precipitates or aggregates were noted.

METHOD OF APPLICATION
Standard plate incorporation assay:
0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of S9-mix (experiments with activation) and 0.1 mL of test solution, positive control or the solvent as negative control. The mixture was poured on minimal agar in Petri dishes.
Preincubation assay:
0.1 mL of the overnight cultures were mixed with 0.5 mL of S9-mix (experiments with activation) and 0.1 mL of test solution, positive control or the solvent as negative control. The mixture was incubated for 30 minutes at 37°C. Thereafter 2 mL of top agar were added to the mixture, which was then poured on minimal agar in Petri dishes.

Each Petri dish contained about 20.0 mL of minimal agar; the top agar was composed of 0.6% agar and 0.6% NaCl.
In the Salmonella experiment, the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM d-biotin dissolved in water.
In the experiment with E. coli, the top agar was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.

RANGE FINDER
The range finding test was carried out with TA 100 and E. coli WP2 uvrA with and without S9-mix at six concentrations of the test substance and one negative control; the highest concentration applied was 5000 µg/plate. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

MAIN MUTAGENICITY TEST
The main mutagenicity test was performed with all strains mentioned above with and without S9-mix. Each of the five selected concentrations of the test substance, a negative and a positive control, were tested using three plates per test concentration. The highest concentration applied had been determined in the preliminary range finder. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

CONFIRMATORY TEST
The conduct of the confirmatory was similar to that of the main test described above.

COLONY COUNTING AND SCORING OF THE PLATES
Colonies were counted electronically (Artek Colony Counter), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The means for all mutagenicity assays were calculated.

Evaluation criteria:

Generally a concentration-related effect should be demonstrable. The test chemical is considered positive if the following criteria are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537, E. coli WP2 uvrA.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains TA 100 or TA 102.

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase in the incidence of revertants was observed in any of the Salmonella strains tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In fact, a growth inhibiting effect in absence of S9-mix was reported for strain TA 98 since the number of revertants was weakly reduced at the concentration of 5000 µg/plate; this effect was not observed with the other strains with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

other: see solvent control

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase in the incidence of revertants was observed.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: see solvent control

Positive controls validity:

valid

Additional information on results:

RANGE-FINDER
Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. At the concentrations of 185.2 µg/plate and above the test substance precipitated on the surface of the agar plates. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

CONFIRMATORY TEST
In the confirmatory experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and WP2 uvrA with the test material, no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control.

PRECIPITATION
At the concentrations of 625 µg/plate and above the test substance precipitated on the surface of the agar plates.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

MAIN MUTAGENICITY TEST – SUMMARY OF RESULTS:

Main Mutagenicity Test without S9-mix
Concentration (µg/plate)	Tester Strain
	TA100	TA1535	TA98	TA1537	TA102	E. coli WP2 uvra
Solvent control	105.33	15.33	25.33	11.67	176	17
312.5	118.33	17	28.33	12	162.33	16
625	104.33	13	25.67	12.33	144.33	19
1250	107.33	13	26	13	124	18.33
2500	115.33	15.67	18.67	12	128.33	15.67
5000	105.33	16.67	10.67	9	126.33	17.33
Positive control	817	729.67	1042.33	1409	781.67	821.67
Main Mutagenicity Test with S9-mix
Concentration (µg/plate)	Tester Strain
	TA100	TA1535	TA98	TA1537	TA102	E. coli WP2 uvra
Solvent control	147	14	52.33	8.33	272.33	25.67
312.5	135.67	15.67	45	10.33	281.33	26.67
625	136	13.33	36.33	11	251	26.33
1250	133.67	17.33	42.67	11.67	238.67	23.33
2500	140.67	9.67	48.67	9.33	212	25
5000	124.33	11.67	40	11	191.33	22.67
Positive control	1243.67	264.67	1161	163.67	949	646