Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 03,2002 to November 11,2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Acid Blue 025:1 - Similar Substance 03
IUPAC Name:
Acid Blue 025:1 - Similar Substance 03
Test material form:
not specified

Method

Target gene:
Strains Genotype Tvpe of mutations indicated

TA 1537 his C 3076; rfa-; uvrB- frame shift mutations
TA 98 his D 3052; rfa-; uvrB-; R-factor frame shift mutations
TA 1535 his G 46; rfa-; uvrB- base-pair substitutions
TA100 his G 46; rfa-; uvrB-; R-factor base-pair substitutions
WP2 uvrA trp-; uvrA- base-pair substitutions and others
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Selective Agar
The plates with the selective agar (minimal agar plates) were made in-house. 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 agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose was delivered from Merck, Germany [D(+)glucose, anhydrous]. The Vogel-Bonner Medium E was prepared in-house.

Overlay Agar
The overlay agat contained per litre:
6.0 g GIBCO BRL Select Agar
6.0 g NaCl (Merck, Germany)
Sterilisation was performed at 121 °C in an autoclave, cooled down to 50'C and dispensed into glass bottles. On day of test peformance the agar was molten in a water bath and 10% aliquotes (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.

NUMBER OF REPLICATIONS: For each strain and dose level including the controls, three plates were used

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
To evaluate the toxicity of the test item 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 item. The concentrations applied were 20.6, 61 .7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate. One plate was prepared per test item concentration, negative and positive controls. The plates were inverted and incubated for about 48 hours at 37± 2 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The range finding test is reported separately.
Statistics:
A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Non genotoxic
Executive summary:

Method

The substance 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 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (Sg fraction) as an extrinsic metabolic activation system. The test item was dissolved in DMSO and tested at five concentrations: 312.5, 625, 1250,2500 and 5000 µg/plate.

ln order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay. 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 (reference item).

Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61 .7, 185.2,555.6, 1666.7 and 5000.0 µg/plate.

Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.

From the results obtained the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.

Results

ln the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations.

ln the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations.

ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. Due to a growth inhibiting effect of the test item in the pre-incubation assay on strain TA 1537, the number of revertant colonies was reduced at the concentration of 5000 µg/plate. No similar effect was observed with the other strains. The test item did not precipitate

on the surface of the agar plates. ln the experiments negative (solvent) and positive control (reference items) treatments were

included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The reference items induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

Conclusion

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.