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Toxicological information

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:
1 December 1998 - 5 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: GLP standards for toxicity testing of chemicals that describing the testing facility require for testing new and specific chemical compounds (Precept No.4, Kankiken No. 233, Eisei No. 38, 63 Kikyoku No.823: revised on November 18, 1988)
Qualifier:
according to guideline
Guideline:
other: GLP standards for toxicity testing of chemicals, Ministry of Labour, Japan (Notification No .76, September 1, 1988).
Qualifier:
according to guideline
Guideline:
other: the Guidelines for screening toxicity testing of chemicals describing the partially revised test procedures require for testing new chemical compounds (Kanpoan No. 287, Eisei No. 127: October 31, 1997; Kikyoku No. 2: October 31, 1997)
Qualifier:
according to guideline
Guideline:
other: the Guidelines for the standards of bacterial mutagenicity testing, Ministry of Labour, Japan (Notification No. 67, June 2, 1997).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-270-3
EC Name:
-
Cas Number:
260408-02-4
Molecular formula:
CAS formula: (C12 H10 O4 S . C6 H6 O . Cl5 P . Cl H4 N)x Molecular formula of the reaction products: (C12 H10 N O2 P)n (n=3-15)
IUPAC Name:
439-270-3
Test material form:
solid
Details on test material:
- Appearance: Yellowish-white, semisolid
- Storage condition: At room temperature away from light

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate S9 mix of rat treated with phenobarbital and 5,6-benzoflavone.
- method of preparation of S9 mix: To prepare S9mix, G-6-P and NADH and NADPH were dissolved in distilled water. Then 0.4M MgCl2, 1.65M KCI and phosphate buffer solution were added. The mix was filtered for sterilization and then S9 was added. The S9mix was kept in ice water bath during use. S9-mix contained per 1 mL: 4 µmol NADPH, 4 µmol NADH, 5 µmol glucose-6-phosphate, 100 µmol sodium phosphate buffer pH 7.4; 8 µmol MgCl2 ; 0.33 µmol KCl and 0.1 mL S9.
Test concentrations with justification for top dose:
Pre-incubation assay:
Experiment 1 (without and with S9) TA100, TA1535, TA1537 and TA98 and WP2uvrA: 5, 10, 50, 100 , 500, 1000, 5000,μg/plate
Experiment 2 (without and with S9) TA100, TA1535, TA1537 and TA98 and WP2uvrA: 156.3, 312.5, 625, 1250, 2500, 5000 μg/plate

The top dose was the highest dose required to be tested.
Vehicle / solvent:
Solvent: dimethylsulphoxide
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide in DMSO 0.1 μg/plate, 0.01 μg/plate, 0.01 μg/plate for TA98, TA100 and WP2uvrA, resp.
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA) in DMSO, 0.5 μg/plate for TA98, 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA
Remarks:
With S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate in each strain for the test item and the positive control and in triplicate for the negative contro group. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar; preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Examining the reduction of the bacterial background lawn using a microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.
- Concentration of the test substance resulting in precipitation: 2500 µg/plate and higher
Evaluation criteria:
A result was judged positive (+) when the number of revertant colonies had a 2-fold or greater increase as compared to that in the negative control group, and when the result was reproducible and dependent upon doses of the test substance; and the other cases were judged negative (-). Bacterial growth inhibition was judged positive when the background lawn of the test substance group was sparse or thin as compared to that of the negative control group.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See attachement for detailed results

TEST CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the substance on the plates was observed at 2500 μg/plate and higher in the experiments with and without metabolic activation

The negative and positive control group were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
In an Ames test, performed in accordance with GLP principles, the substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

An Ames performed in accordance with GLP principles. The test item was tested up to concentrations of 5000 μg/plate with and without metabolic activation in duplicate in two independent experiments (pre-incubation). Precipitation of the substance was observed at 2500 μg/plate and higher in the experiments with and without metabolic activation. No cytotoxicity was observed. The negative and positive control group were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not show a 2-fold or greater increase as compared to that in the negative control group in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in both experiments. In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.