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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:
From November 15 to December 20, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Acid Violet 048
IUPAC Name:
Acid Violet 048

Method

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):
CELLS USED
- Source of cells: the histidine-auxotrophic strains of Salmonella typhimurium TA 98 and TA 1535 were obtained from Prof. B. Ames, Berkeley, USA (1983). Strain TA 100 was obtained from Dr. M. Schupbach, Hoftmann-La Roche Limited, Basel, Switzerland (1986). Strain TA 1537 was obtained from Dr. S. Albeitini, Hoftmann-La Roche Limited, Basel, Switzerland (1994).

STORAGE
The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 + 1 ml) in a deep freezer at about -80 °C.

PRECULTURES
60 µl aliquots from a frozen stock were grown in 60 ml nutrient broth medium in a 300 ml Erlenmeyer flask (Nutrient broth No. 2, Oxoid Ltd. Basingstoke, U.K) for 8 hours by orbital shaking at 37 °C, 130-140 rounds per minute and then used for the experiment. The bacterial cultures were incubated in a temperature / time controlled incubator.

MEDIA USED
- Periodically checked for characteristics of the strains: the characteristics of the strains were checked every two to six months.
- Periodically checked for ampicillin resistance: yes.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: the tryptophan auxotrophic strain of Escherichia coli (WP2 uvrA) was obtained from the National Collection of industrial Bacteria, Aberdeen, Scotland (1977).

STORAGE
The strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10 + 1 ml) in a deep freezer at about -80 °C.

PRECULTURES
60 µl aliquots from a frozen stock were grown in 60 ml nutrient broth medium in a 300 ml Erlenmeyer flask (Nutrient broth No. 2, Oxoid Ltd. Basingstoke, U.K) for 8 hours by orbital shaking at 37 °C, 130-140 rounds per minute and then used for the experiment. The bacterial cultures were incubated in a temperature / time controlled incubator.

MEDIA USED
- Periodically checked for characteristics of the strains: the characteristics of the strains were checked every two to six months.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver post motochondrial supernatant
Test concentrations with justification for top dose:
MAIN EXPERIMENTS: 312.5, 625, 1250, 2500 and 5000 µg/plate, with and without S9 mix
PRELIMINARY TEST: 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate, with and without S9 mix
Vehicle / solvent:
- Solvent: DMSO
- Justification for choice of solvent: test item was soluble in DMSO up to the concentration of 50 mg/ml. No aggregates or precipitation were noted.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
other: 2-aminoanthracene
Details on test system and experimental conditions:
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 \/ogel-Bonner Medium E was prepared in-house.

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

EXPERIMENTAL PERFROMANCE
The following materials were mixed in a test tube and poured onto the minimal agar plates:
- 100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
- 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution butter (tor test without metabolic activation);
- 100 µl Bacteria suspension (ct. test system, pre-culture oi the strains);
- 2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37 °C for 30 minutes. After pre-incubatior 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. ln addition, the respective controls (solvent) and positive controls were run together with each strain.
After solidification the plates were incubated upside down for at least 48 hours at 37 ± 2 °C in the dark.

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

COUNT OF COLONTY
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting.
Observations indicating precipitation oi the test item in the overlay agar or a reduced or absent bacterial background lawn were registered additionally.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a range finding test was carried out with strains Styphimuriurn TA 100 and E. coll 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 µg/plate. One plate was prepared per test item concentration, negative and positive control. 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.

MAMMALIAN MICROSOMAL FRACTION
S9 (Preparation in-house)
Rat-liver post mitochondrial supernatant (S9 traction) was prepared in advance from male rats [HanBrl:WlST (SPF)], delivered by testing facility. The animals were treated with Aroclor 1254, 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content oi the S9 traction was 42.5 mg/ml.

Mix
On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP, in 100 mM sodium phosphatebuffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Arnes et al.

ACCEPTABILITY ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges listed in the historical data and if the results of the positive controls meet the criteria for a positive response. Normal bacterial background lawn shall be visible in the solvent control.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98) TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number oi revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative controls (solvent) such an increase is not considered biologically relevant.
A reduction in the number of revertant colonies in the groups treated with the test item by > 50 %, compared with the respective solvent control, is reported as toxic effect.
Statistics:
A statistical analysis was not required.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation ot the test item was visible.
ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. Toxic effects, evident as a reduction in the number of revenants, were observed in the first experiment without activation in strain TA 100 at the concentrations of 2500 and 5000 µg/plate and in strains TA 1535, TA 98, TA 1537, WP2 uvrA at the concentration of 5000 µg/plate.
In the experiment with activation, similar effects occurred in strain TA 100, TA 98, WP2 uvrA at the concentration of 5000 µg/plate and in strain TA 1537 at the concentrations of 2500 and 5000 µg/plate.
In the second experiment without activation, a reduction in the number of revenants was visible in strain TA 100 at the concentrations ot 2500 and 5000 µg/plate and in strain TA 1587 at the concentration of 5000 µg/plate. Similar ettects occurred in the experiment with activation in strains TA 100, TA 98 and TA 1537 at the concentration of 5000 µg/plate. No precipitation of the test item was seen on the surface of the agar plates.
No substantial increase in revenant colony numbers of any of the five tester strains was observed following treatment with test item at any concentration level, neither in the presence nor in the absence of a metabolic activation system (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

CONTROLS
Appropriate reterence mutagens were used as positive controls. They showed a distinct increase of induced reveitant colonies.

PRELIMINARY TEST
Since no relevant toxic effects were observed, 5000 µg/plate were chosen as maximal concentration.

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or trameshitts in the genome of the strains used.
Executive summary:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimuritim 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 (S9 fraction) as an extrinsic metabolic activation system. Each concentration, the negative and the positive controls were tested in triplicate. The test item was dissolved in DMSO and tested at five concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate.

In order to confirm the results, the experiment was 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.

Previously, a pre-experiment for toxicity (range finding test) was performed with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The concentrations of 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate were tested with and without metabolic activation. Normal background growth was observed with both strains. Due to a growth inhibiting effect of the test item on strain TA 100 without metabolic activation, the number of revertant colonies was reduced at the concentration of 5000 µg/plate. No precipitation of the test item was seen 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. In the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with the test item at any concentrations.

In 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.

In both mutagenicity tests normal background growth was observed with all strains at all concentrations. Toxic effects, evident as a reduction in the number of reveitants, were observed in the first experiment without activation in strain TA 100 at the concentrations of 2500 and 5000 µg/plate and in strains TA 1535, TA 98, TA 1537, WP2 uvrA at the concentration of 5000 µg/plate. In the experiment with activation, similar effects occurred in strain TA 100, TA 98, WP2 uvrA at the concentration of 5000 µg/plate and in strain TA 1537 at the concentrations of 2500 and 5000 µg/plate. ln the second experiment without activation, a reduction in the number of revertants was visible in strain TA 100 at the concentrations of 2500 and 5000 µg/plate and in strain TA 1587 at the concentration of 5000 µg/plate. Similar effects occurred in the experiment with activation in strains TA 100, TA 98 and TA 1537 at the concentration of 5000 µg/ptate. No precipitation of the test item was seen on the surface of the agar plates.

In the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to he within acceptable ranges, The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain tunctioning and the activity of the S9-mix.

Conclusion

In 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 trameshitts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in this Salmonella typhirnurium and Escherichia coli reverse mutation assay.