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Toxicological information

Genetic toxicity: in vitro

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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Acceptable, well documented study report which meets basic scientific principles. The fith strain (E. coli) was also tested. Deficiencies: non-GLP, repeat experiments not performed for all strains but as shown in the supporting study, the result is reducable and no contradictory results occurred.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 4 concentrations tested)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Expiration date of the lot/batch: July 1996

Method

Target gene:
his- / trp-gene
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
- Range in the cytotoxicity test: 20.57 - 5000 µg/plate;
- Range in the mutagenicity test: with/without metabolic activation: 625 - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: bidistilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below: details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

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 the activation mixture (Rat liver S9 fraction: 100 µl/ml, NADP: 4 µmol/ml, MgCl2: 8 µmol/ml, KCl: 33 µmol/ml, Na-phosphate-buffer, pH 7.4: 100 µmol/ml, Glucose-6-phosphate 5 µmol/ml; experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water. 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. Colonies were counted electronically with an Artek counter.

Positive controls (without metabolic activation):

- TA 100: sodium azide in bidist. water (5.0 µg/plate);
- TA 1535: sodium azide in bidist. water (5.0 µg/plate);
- E.coli WP2 uvrA: 4-nitroquinoline-N-oxide in DMSO (2.0 µg/plate);
- TA 98: 2-nitrofluorene in DMSO (20.0 µg/plate);
- TA 1537: 9-aminoacridine in DMSO (150.0 µg/plate);

Positive controls (with metabolic activation):

- TA 100: 2-aminoanthracene in DMSO (2.5 µg/plate);
- TA 1535: cyclophosphamide*H2O in bidist. water (400.0 µg/plate);
- E. coli WP2 uvrA: 2-aminoanthracene in DMSO (50.0 µg/plate);
- TA 98: 2-aminoanthracene in DMSO (2.5 µg/plate);
- TA 1537: 2-aminoanthracene in DMSO (2.5 µg/plate);

Evaluation criteria:
Assay acceptance criteria:

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response:

The test substance is considered to be mutagenic in this test system if:
A positive effect is observed in one strain and the effect can be reproduced in a confirmatory experiment. A positive effect is observed in two or more strains. A positive effect is defined as an increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 2.0 with strains TA 98, TA 1535, TA 1537 and WP2 uvrA, or by a factor of at least 1.5 with strain TA 100. Generally a concentration-related effect should be demonstrable. If equivocal results are obtained, the final decision has to be based on scientific judgement.
Statistics:
Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Results and discussion

Test results
Species / strain:
other:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: not observed


RANGE-FINDING/SCREENING STUDIES:
The concentration range of TK 11831 to be tested in the mutagenicity test was determined in a preliminary toxicity test. The toxicity test was carried out with strains S. typhimurium TA 100 and E.coli WP2 uvrA without and with metabolic activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA: negative and positive historical control data and acceptable ranges for negative controls were available


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 13 1.31 (1250) no negative 107.62 (2-nitrofluorene)
  yes 28.5 1.07 (625 / 5000) no negative 30.63 (2-aminoanthracene)
TA 100 no 113 0.95 (5000) no negative 8.92 (sodium azide)
  yes 129.5 0.96 (5000) no negative 5.75 (2-aminoanthracene)
TA 1537 no 8 0.88 (1250) no negative 218.50 (9-aminoacridine)
  yes 5.5 1.64 (1250) no negative 16 (2-aminoanthracene)
TA 1535 no 15 1.03 (5000) no negative 61.47 (sodium azide)
  yes 13 1.31 (5000) no negative 31.19 (cyclophosphamide)
E. coli WP2 uvrA no 21 1.21 (2500) no negative 30.4 (4-NQO)
yes 30 0.83 (1250) no negative 45.87 (2-aminoanthracene)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative