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Genetic toxicity: in vitro

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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

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,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) 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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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 resultsopen allclose all
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
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
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
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
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
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.