O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

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:

April 19, 1989 - May 29, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

(no statistics)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

EC Number:

406-940-1

EC Name:

O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

Cas Number:

126019-82-7

Molecular formula:

C30-54H47-87O3PS

IUPAC Name:

O-2-(2-methylnonyl)phenyl O-4-(2-methylnonyl)phenyl O-2-(2-methyloctyl)phenyl phosphorothioate

Details on test material:

- Physical state: viscous liquid, insoluble in water; pH 7
- Storage condition of test material: room temperature

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Method

Target gene:

Salmonella typhimurium: His (-/-)
E. Coli: Tryp (-/-)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentrations in the main test (with metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentrations in the main test (without metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentration range in the preliminary toxicity test: 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/0.1 mL

Vehicle / solvent:

Acetone (test article); DMSO (positive controls)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with and without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION (in agar, plate incorporation):

DURATION
- Incubation period: about 48 h at 37 ± 1.5°C in darkness

NUMBER OF REPLICATIONS:
- 3 Petri dishes per strain and per group per experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration
level for one or more of the following strains: TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA,
- 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 strain TA 100. Generally a concentration-related effect should be demonstrable

Statistics:

not performed

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

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 6

Key result

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at the concentrations of 500 µg/0.1 mL and above the test substance precipitated in soft agar

RANGE-FINDING/SCREENING STUDIES:
Nine concentrations ranging from 20 to 5000 ug/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 ug/0.1 mL.

Any other information on results incl. tables

Experiment I

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2uvrA
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	13	28	117	153	8	13	4	17	7	19	22	29
5	17	32	118	149	16	13	7	14	10	16	24	28
10	14	29	114	146	9	3	3	13	9	17	18	26
50	24	36	128	136	14	16	6	18	5	21	19	17
100	16	23	114	141	16	13	6	14	5	16	17	19
500	19	29	124	153	14	11	9	16	5	16	18	24
1000	15	32	126	137	15	13	7	15	8	18	19	26
5000	15	27	120	136	13	12	4	13	9	18	23	27
positive controls:
solvent control	14	23	100	139	8	14	7	14	8	19	17	21
concentration A	139	385	481	428	199	163	21	127	200	140	362	609
concentration B	222	376	754	542	313	94	149	124	358	123	674	718

Experiment II

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2uvrA
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	17	28	135	185	14	9	6	10	7	20	24	34
5	15	26	118	186	15	10	4	18	5	23	21	33
10	11	28	120	188	10	9	8	17	3	15	27	27
50	22	27	105	156	9	8	6	11	11	14	25	22
100	14	21	108	145	19	8	5	17	5	15	19	29
500	23	29	124	133	9	11	8	14	11	16	25	24
1000	14	29	113	153	14	9	3	14	5	19	23	28
5000	20	24	123	133	17	12	5	15	9	19	24	35
positive controls:
solvent control	13	25	104	135	11	12	3	16	6	22	18	24
concentration A	137	455	318	289	219	144	30	191	189	179	392	498
concentration B	268	429	388	461	410	160	171	140	265	157	562	441

The slight increase in the number of back-mutant colonies observed with strain TA 1535 in the first experiment without microsomal activation at the concentrations of 5 and 100 µg/0.1 ml could not be confirmed in the second experiment and is therefore attributed to spontaneously occurring back-mutants.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli WP2UvrA in two independent experiments. These tests were following GLP guidelines and were based on the OECD testing guideline 471. In a dose range finding test, the test article was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. At this dose level no toxicity was observed. In the mutation assays, the test substance was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. The test article precipitated at concentrations of 500 µg/0.1 ml and above. No decrease in the number of revertants was observed. No dose-related increase in the number of revertant both in the absence and presence of S9-metabolic activation was observed. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.