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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:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
uvrA and uvrB
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Controls of the genotype of the strains:
The characteristics of the strains were checked every two months. Histitidine-auxtorophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA98 and TA100) were additionally checked for ampicillin resistance. The tryptophan-auxotrophy of E. coli WP2uvrA was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light. Furthermore, all strains were checked for their reversion properties with known mutagens (positive controls).
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction).
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Controls of the genotype of the strains:
The characteristics of the strains were checked every two months. Histitidine-auxtorophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA98 and TA100) were additionally checked for ampicillin resistance. The tryptophan-auxotrophy of E. coli WP2uvrA was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light. Furthermore, all strains were checked for their reversion properties with known mutagens (positive controls).
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction).
Test concentrations with justification for top dose:
Concentration range in the range-finding test:
20.6 to 5000.0 µg/plate

Concentration ranges in the Mutagenicity Tests
Original experiment: 312.5 to 5000.0 µg/plate

Confirmatory experiment with activation:
S. typhimurium: 78.1 to 1250.0 µg/plate
E.coli WP2 uvrA: 312.5 to 5000.0 µg/plate

Confirmatory experiment without activation:
S. typhimurium TA 100, TA 102, TA 98, TA 1537: 32.5 to 1000.0 µg/plate
S. typhimurium TA 1535 and E. coli WP2 uvrA: 312.5 to 5000.0 µg/plate
Vehicle:
Dimethylsulphoxide (DMSO)
Controls
Negative controls:
yes
Remarks:
the solvent alone was used as a negative control
Solvent controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoanthracene, 4-Nitroquinoline, Aroclor 1254, Dimethylsulphoxide
Details on test system and conditions:

Solubilisation of the test substance:
CA 2219 A was dissolved on DMSO at room temperature. The test substance was soluble up to the concentration of 50 mg/L. Lower concentrations of the test substance were obtained by serial dilution of the stock solution with DMSO. No precipitates or aggregates were noted.

Setting up of the plates:
Standard plate incorporation assay: 0.1ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (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.
Preincubation assay: 0.1 ml of the overnight cultures were mixed with 0.5ml of the activation mixture (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 incubated for 30 min. at 37°C. Therafter 2 ml of top agar was added to the mixtures and they were poured on minimal agar in Petri dishes.
Each Petri dish contained about 20.0 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 0.5mM d-biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5mM L-tryptophan dissolved in water.

Range finding tests:
A range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without 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 three. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Negative and positive controls
The solvent alone was used as the negative control.
The positive controls (reference mutagens) with and without metabolic activation can be found in Table 1 and Table 2.
Evaluation criteria:

Scoring of the plates
Colonies were counted electronically using Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong colouration of the agar plates might have interfered with automatic counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally.

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response
The test substance will be considered to be positive in the 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 for one or more of the following strains:
TA 98, TA 1535, TA 1537, E.coli WP2 uvrA.
• 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 strains TA 100 or TA 102.

Generally a concentration-related effect should be demonstrable.
Statistics:
A statistical analysis was not performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:

Range finding tests:
Six concentrations of CA 2219 A ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 and the strain Escherichia coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation.
Due to a growth inhibiting effect of the test substance in the experiment with metabolic activation on strain TA 100, the number of revertant colonies was reduced at the upper concentrations.
The background growth was invisible at the highest concentration.
In the experiment without activation and on strains TA 100 and E. coli a similar effect occurred at the highest concentration. The background growth was reduced at the highest concentration.
At the concentrations of 5000.0 µg/plate the test substance precipitated in soft agar
From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 312.5 to 5000.0 µg/plate with and without metabolic activation.

Mutagenicity Tests:
In the original and in the performed with and without metabolic activation, treatment of strains TA98, TA 100, TA 102, TA 1535, TA 1537 and WP2 uvrA with CA 2219 A did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants in comparison with the negative control.

In the confirmatory experiments performed out with and without metabolic activation, again after treatment of strains TA98, TA100, TA102, TA1535, TA 1537 and WP2uvrA with CA 2219 A, no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control.

Due to growth inhibiting effect of the test substance a reduction in the number of revertant colonies was visible in the experiments with metabolic activation on all strains of Salmonella tyhimurium at the concentrations of 1250 to 5000 0 µg/plate. In the experiments without activation a reduction in the number of revertant colonies on strains TA 100, TA 102 (1000 to 5000 0 µg/plate), TA 1537 (2500 and 5000 0 µg/plate), TA 98 and E.coli (5000 0 µg/plate).
The growth of the bacterial background lawn was occasionally reduced or invisible at higher concentrations.
At the concentration of 5000 0 µg/plate, the test substance precipitated on the surface of the agar plates.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the result of these experiments and on standard evaluation criteria, it is concluded that CA 2219 A (Intermediate of CGA 276854) and it’s metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.
Executive summary:

CA 2219 A (Intermediate of CGA 276854), identified as a brown liquid with a purity of 95.5%, batch no. 249-GE001/SU, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochrondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in a range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system in the original mutagenicity test. In order to confirm the results, the experiment with metabolic activation was repeated on the strain of S. typhimurium at 78.1 to 1250 µg/plate and E. coli at 312.5 to 5000.0 µg/plate. In the repeat experiment without activation on strains TA 98, TA 100, TA 102, TA 1537 the concentrations of 62.5 to 1000.0 µg/plate were tested. The strains TA 1535 and E. coli were tested with the concentrations of 312.5 to 5000.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. 

The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The confirmatory experiment with metabolic activation was carried out as preincubation assay. 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of CA 2219 A led to an increase in the incidence of either histidine- or tryptophan- prototrophic mutants by comparison with the negative control. 

Based on the result of these experiments and on standard evaluation criteria, it is concluded that CA 2219 A (Intermediate of CGA 276854) and it’s metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.