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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:
1998-02-23 to 1998-03-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
2-aminoanthracene was used as the sole indicator of the efficacy of the S9 mix. Only 4 analysable results due to cytotoxicity in TA100, TA1535 and TA1537.
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
-Appearance: colorless or light yellow
-Molecular weight: 136.1
-Water solubility at 25 °C: 98 mg/ 100 mL
-Soluble in dimethylsulfoxide, acetone, almost insoluble in sesame oil
-Impurities: 0.41 % benzoic acid, 0.11 % o-toluic acid, 0.69 % other low boiling fractions
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated in the dark

Method

Target gene:
TA98: hisD
TA100: hisG
TA1535: hisG
TA1537: hisC
WP2uvrA: trpE
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB, rfa
Remarks:
TA98 and TA100: pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (containing 10 % S9, 8 µM MgCl2 x 6 H2O, 33 µM KCl, 5 µM D-Glucose-6-phosphat, 4 µM NADPH, 4µM NADH, 100 µM sodium buffer (pH 7.4), sterile water))
Test concentrations with justification for top dose:
A preliminary test (preincubation, with and without metabolic activation) was conducted for purpose of dose range finding with the following concentrations: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.
The following concentrations were selected for the main tests (preincubation method, without metabolic activation): 156, 313, 625, 1250, 2500 and 5000 µg/plate and (preincubation method, with metabolic activation): 313, 625, 1250, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in water at a concentration of 50 mg/mL. Thus, the test substance was dissolved in DMSO and diluted with DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
furylfuramide
other: 2-aminoanthracene
Remarks:
positive control substances were dissolved in DMSO, except sodium azide (dissolved in water for injection)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

MAIN EXPERIMENT (two independent experiments with and without metabolic activation)
The test item was preincubated with the test strain (containing approximately 10^9 viable cells/mL) and sterile buffer (0.5 mL of 0.1 M sodium phosphate buffer, pH 7.4) or the metabolic activation system (0.5 mL of S9 mix) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate.
0.1 mL of test item (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker.
The plates were incubated at 37°C for 48 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter or visually.

NUMBER OF REPLICATIONS: 3 replicates for each experimental point in the main experiment, one sample in the preliminary test

DETERMINATION OF CYTOTOXICITY
Prior to the main test a preliminary test (preincubation method, with and without metabolic activation) was performed in the test strains TA98, TA100, TA1535, TA1537 and WP2uvrA. The following concentrations were tested: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.
Rationale for test conditions:
The test concentrations were chosen based on a preliminary test. Cytotoxicity was observed at 5000 µg/plate (with and without metabolic activation). Precipitation was observed for a concentration of 5000 µg/plate (with metabolic activation). No mutagenicity was observed.
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is increased compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and WP2uvrA
- positive results have to be reproducible
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
no statistical method was used

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537, and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results"
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results"
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- test item precipitation was noted at a concentration 5000 μg/plate in the presence of S9 mix in all test strains.
- cytotoxicity was observed at a concentration 5000 μg/plate in the absence of S9 mix in all test strains.

MAIN STUDY (2 independent experiments)
1) Test-specific confounding factors:
- Precipitation: test item precipitation was noted in both experiments with metabolic activation, at a concentration of 5000 μg/plate in all test strains.

2) Cytotoxicity:
- cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments with metabolic activation in all test strains at 5000 μg/plate and in test strains TA100, TA1535 and TA1537 at 2500 µg/plate.

3) Genotoxicity:
- no increase in revertant colony numbers as compared with control counts was observed for the test item in any of the two independent experiments without and with metabolic activation.
- positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not considered to have a mutagenic potential.