Registration Dossier

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
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Acid Red 337

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver and S9 mix from hamster liver
Test concentrations with justification for top dose:
Test groups
first plate incorporation test:
with (10% rat liver) and without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate

second plate incorporation test:
without metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate (TA 100 and TA 1535)
1.6, 5, 16, 50, 160 and 500 µg/plate (TA 1537 and TA 98)

preincubation test:
without metabolic activation:
0.05, 0.16, 0.5, 1.6, 5 and 16 µg/plate (TA 100)
0.5, 1.6, 5, 16, 50 and 160 µg/plate (TA 1535)
0.16, 0.5, 1.6, 5, 16, 50, 160 and 500 µg/plate (TA 1537)
1.6, 5, 16, 50, 160 and 500 µg/plate (TA 98)
50, 160, 500, 1600 and 5000 µg/plate (WP2uvrA)

with metabolic activation (30 % Syrian golden hamster liver):
1.6, 5, 16, 50, 160 and 500 µg/plate (TA 100 and TA 1535
5, 16, 50, 160, 500 and 1600 µg/plate (TA 1537)
5, 16, 50, 160, 500, 1000 and 1600 µg/plate (TA 98)
50, 160, 500, 1600 and 5000 µg/plate (WP2uvrA)

repeat preincubation test:
without metabolic activation:
0.5, 1.6, 2.5, 5, 10, 16 and 50 µg/plate (TA 1537)
with metabolic activation (30 % Syrian golden hamster liver):
160, 250, 500, 650, 800 and 1000 µg/plate (TA 98)


Control groups
negative controls:
a: untreated control
b: solvent controls (0 µg/plate)

positive controls:
a: without metabolic activation:
sodium-azide for strain TA 100 and TA 1535
9-aminoacridine for strain TA 1537
2-nitrofluorene for strain TA 98
4-nitroquinoline-N-oxide for strain WP2uvrA
b: with metabolic activation (10% rat liver): 2-aminoanthracene for all tester strains
c: with metabolic activation (30% Syrian golden hamster liver and preincubation):
2-aminoanthracene for strain TA 100, TA 1535, TA 1537 and WP2uvrA
congo red for strain TA 98
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubitiy in water is not sufficient
- Formulation: suspended in DMSO at appropriate concentrations immediately before use
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
congo red
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Source of bacteria: stock cultures in the bank of "Genetic Toxicology", HMR Deutschland GmbH, ProTox, prepared from the original bacterial strains
Test organisms: Salmonella typhimurium strains
TA 98 hisD3052 rfa uvrB +R,
TA 100 hisG46 rfa uvrB +R,
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
and
E. coli WP2 uvrA pKM101
Experimental conditions in vitro: approx. 37°C in an incubator

Two independent mutation tests were performed unless clearly positive or dose-related activity was observed in the first test. Where results were negative or equivocal, a second test was conducted. This included a pre-incubation step if the first test was clearly negative. Pre-incubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The highest concentration in the first mutation experiment was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used. Suitable dose levels used in the second experiment may be different depending on any toxicity seen in the first experiment. A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators.
Thinning of the bacterial lawn was evaluated microscopically.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48°C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in deionized water)
In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 30 minutes at approx. 30 DC and the top agar composition as described was added accordingly.
After mixing, and pre-incubation if appropriate, the liqUid was poured into a petri dish containing a 25, mllayer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37°C in the dark, colonies (his+ and trp+ revertants) were counted by hand or by a suitable automatic colony counter.
The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for the counter's sensitiveness.
Evaluation criteria:
Criteria for a valid assay
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Sterility checks and control plates
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Solubility and toxicity
The test compound was dissolved in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in the plate incorporation test.
Visible precipitation of the test compound on the plates was observed at 5000 µg/plate.
In the plate incorporation test dose-related toxicity was observed at concentrations of 1600 µg/plate and above in the presence of metabolic activation with the tester strains TA 100, TA 1535 and TA 1537, and at 5000 µg/plate with the strain TA 98.
In the absence of metabolic activation, toxicity was observed at concentrations of 500 µg/plate and greater (TA 100, TA 1535, TA 1537), at 1600 µg/plate and greater (TA 98).
In this mutagenicity experiment toxicity was not observed with and without S9-mix to the bacterial strain Escherichia coli WP2uvrA.
Thinning of bacterial lawns and in most cases also a reduction in the number of colonies were observed at these dose levels.
The top dose level for each bacterial strain in the second experiment was selected on the basis of the first mutation experiment results.
In the preincubation test toxicity was observed without metabolic activation in a dose range of 500 to 5000 µg/plate.
In the presence of metabolic activation the test compound proved to be toxic to the bacterial strains TA 1535 and WP2uvrA in a dose range of 1600 to 5000 µg/plate.

Mutagenicity
In both independent mutation tests, Acid Red 337 was tested for mutagenicity with the same concentrations as described under “Solubility and toxicity”. The number of colonies per plate with each strain as well as mean values of 3 plates are given.
Plate incorporation test:
The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.
Preincubation test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described.
All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Applicant's summary and conclusion

Conclusions:
Acid Red 337 is not mutagenic in the standard plate test (plate incorporation test) or in the preincubation method according to Prival
Executive summary:

Acid Red 337 (Batch No. DYWB 827) was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA.

Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the compound was dissolved in DMSO, and each bacterial strain was exposed to 5 dose levels in the plate incorporation test, and to 5 or 6 dose levels in the preincubation test. Doses for the plate incorporation test ranged from 50 to 5000 µg/plate. Dose ranges for the preincubation test were variable across bacterial strains to account for varying susceptibilities to cytotoxic effects: low doses ranged from 5 to 1600 µg/plate, and high doses ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: In the plate incorporation test toxicity was observed without metabolic activation in a dose range of 500 to 1600 µg/plate and above to the Salmonella typhimurium bacterial strains. In the presence of metabolic activation, the test compound proved to be toxic to the Salmonella typhimurium bacterial strains at concentrations of 1600 µg/plate and above. In the absence and in the presence of metabolic activation the test compound proved to be not toxic to the bacterial strain Escherichia coli WP2uvrA.
In the preincubation test, toxicity was observed without metabolic activation in a dose range of 500 to 5000 µg/plate and above.
In the presence of hamster liver metabolic activation, the test compound proved to be toxic to the bacterial strains TA 1535 and WP2uvrA in a dose range of 1600 to 5000 µg/plate and above.

Visible precipitation of the test compound on the plates was observed at 5000 µg/plate.

Plate incorporation test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with Acid Red 337 did not result in relevant increases in the number of revertant colonies.

Preincubation test:

In the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival Acid Red 337 did not result in relevantincreases in the number of revertant colonies with any of the tester strains.

Summarizing, it can be stated that Acid Red 337 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.