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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)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1987)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TKA 40135 (CGI 819)

Method

Target gene:
histidine or tryptophan auxotrophy

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: defect in the excision repair system (uvrB) , presence of rfa character
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: defect in the excision repair system (uvrB) , presence of rfa character, AT base pair at the primary reversion site
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: AT base pair at the primary reversion site , DNA repair-proficient
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction obtained from the liver of male sprague-dawley rats treated with AROCLOR 1254, mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)
Test concentrations with justification for top dose:
Range-finding test: 20.6 - 5000 µg/plate
Main test: 312.5 - 5000 µg/plate
Confirmation: 312.5 - 5000 µg/plate
Vehicle / solvent:
DMSO was used as vehicle since the test substance was soluble up to the concentration of 50 mg/mL in this vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
see solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
in DMSO at 1.5 µg/plate in TA 100, TA 1537, TA 98; in DMSO at 5.0 µg/plate in TA 102; in DMSO at 20 µg/plate in E. coli
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
cyclophosphamide
Remarks:
in bidistilled water at 200 µg/plate in TA 1535
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
sodium azide
Remarks:
in bidistilled water at 2 µg/plate in TA 100 and TA 1535
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: 4-Nitroquinoline
Remarks:
in DMSO at 2 µg/plate in E. coli
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
mitomycin C
Remarks:
in bidistilled water at 0.5 µg/plate in TA 102
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
2-nitrofluorene
Remarks:
in DMSO at 5 µg/plate in TA 98
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
9-aminoacridine
Remarks:
in DMSO at 80 µg/plate in TA 1537
Details on test system and experimental conditions:
PREPARATION OF THE TEST SOLUTIONS
The test material was dissolved in the dark in DMSO after slightly warmed up. The test substance was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. The test
substance and the solutions were handled in darkness unter red light. No precipitates or aggregates were noted.

METHOD OF APPLICATION
Standard plate incorporation assay:
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 S9-mix (experiments with activation) and 0.1 mL of test solution, positive control or the solvent as negative control. The mixture was poured on minimal agar in Petri dishes.
Preincubation assay:
0.1 mL of the overnight cultures were mixed with 0.5 mL of S9-mix (experiments with activation) and 0.1 mL of test solution, positive control or the solvent as negative control. The mixture was incubated for 30 minutes at 37°C. Thereafter 2 mL of top agar were added to the mixture, which was then poured on minimal agar in Petri dishes.

Each Petri dish contained about 20.0 mL of minimal agar; the top agar was composed of 0.6% agar and 0.6% NaCl.
In the Salmonella experiment, the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM d-biotin dissolved in water.
In the experiment with E. coli, the top agar was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.

RANGE FINDER
The range finding test was carried out with TA 100 and E. coli WP2 uvrA with and without S9-mix at six concentrations of the test substance and one negative control; the highest concentration applied was 5000 µg/plate. 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.

MAIN MUTAGENICITY TEST
The main mutagenicity test was performed with all strains mentioned above with and without S9-mix. Each of the five selected concentrations of the test substance, a negative and a positive control, were tested using three plates per test concentration. The highest concentration applied had been determined in the preliminary range finder. 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.

CONFIRMATORY TEST
The conduct of the confirmatory was similar to that of the main test described above.

COLONY COUNTING AND SCORING OF THE PLATES
Colonies were counted electronically (Artek Colony Counter), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The means for all mutagenicity assays were calculated.

Evaluation criteria:
Generally a concentration-related effect should be demonstrable. The test chemical is considered positive if the following criteria 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.


Statistics:
A statistical analysis was not performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in the incidence of revertants was observed in any of the Salmonella strains tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In fact, a growth inhibiting effect in absence of S9-mix was reported for strain TA 98 since the number of revertants was weakly reduced at the concentration of 5000 µg/plate; this effect was not observed with the other strains with and without S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
other: see solvent control
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in the incidence of revertants was observed.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: see solvent control
Positive controls validity:
valid
Additional information on results:
RANGE-FINDER
Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. At the concentrations of 185.2 µg/plate and above the test substance precipitated on the surface of the agar plates. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

CONFIRMATORY TEST
In the confirmatory experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and WP2 uvrA with the test material, no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control.

PRECIPITATION
At the concentrations of 625 µg/plate and above the test substance precipitated on the surface of the agar plates.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

MAIN MUTAGENICITY TEST – SUMMARY OF RESULTS:

Main Mutagenicity Test without S9-mix     

Concentration (µg/plate)

Tester Strain

TA100

TA1535

TA98

TA1537

TA102

E. coli WP2 uvra

Solvent control

105.33

15.33

25.33

11.67

176

17

312.5

118.33

17

28.33

12

162.33

16

625

104.33

13

25.67

12.33

144.33

19

1250

107.33

13

26

13

124

18.33

2500

115.33

15.67

18.67

12

128.33

15.67

5000

105.33

16.67

10.67

9

126.33

17.33

Positive control

817

729.67

1042.33

1409

781.67

821.67

Main Mutagenicity Test with S9-mix

Concentration (µg/plate)

Tester Strain

TA100

TA1535

TA98

TA1537

TA102

E. coli WP2 uvra

Solvent control

147

14

52.33

8.33

272.33

25.67

312.5

135.67

15.67

45

10.33

281.33

26.67

625

136

13.33

36.33

11

251

26.33

1250

133.67

17.33

42.67

11.67

238.67

23.33

2500

140.67

9.67

48.67

9.33

212

25

5000

124.33

11.67

40

11

191.33

22.67

Positive control

1243.67

264.67

1161

163.67

949

646

Applicant's summary and conclusion