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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 30,2012 from July 06,2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5
Deviations:
no
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
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): SSP-SAMPLE 1

Method

Target gene:
no data
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5000, 2500, 1250, 625, 313, 156, 78.1 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:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The following growth media were used:
Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use.
This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved.
The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL).

DURATION
- Preincubation period:The treatment mixture will be vortexed and placed at 37°C for 30 minutes
- Incubation period: approximately 72 hours at 37°C.
- Preliminary toxicity test:A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: high titre of viable bacteria (1-5 x 10^9 cell/ml)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Sterility check: The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
The data will be presented in tabular form. The individual plate counts for each experiment will be given, together with the means and standard errors of the means, and regression analyses.
Evaluation of Ames test data based on a ‘doubling rate’ has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1981).

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:
not determined
Vehicle controls validity:
not examined
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:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:Moderate precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration.
- Solubility:Solubility of the test item was evaluated in a preliminary trial using DMSO, acetone and ethanol. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. A suspension of the test item, suitable for treatment but not for serial dilution preparation, was found in DMSO at 125 mg/mL after sonication at 37 °C for 10 minutes. A clear solution was obtained in DMSO at 62.5 mg/mL after sonication at 37 °C for 30 minutes. These results permitted a maximum concentration of 5000 g/plate to be used in the toxicity test.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity was observed with all tester strains at higher dose levels, both in the absence and presence of S9 metabolism. A more pronounced toxic effect, with toxicity at all or almost all dose levels, was observed with TA1535 and TA1537 in the absence of S9 metabolism and with TA100 both in the absence and presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a suspension/solution in dimethylsulfoxide (DMSO).

The test item was assayed in the toxicity test at a maximum concentration of 5000 g/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 g/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration. Toxicity was observed at the highest or at the two highest dose levels, with TA1535 and TA1537 in the absence of S9 metabolism and with TA100 both in the absence and presence of S9 metabolism. In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

Tester strain S9 Dose level (µg/plate)
TA1535, WP2 uvrA, TA98 ± 5000, 2500, 1250, 625, 313
TA1537 +
TA100 + 5000, 2500, 1250, 625, 313, 156
TA1537, TA100 2500, 1250, 625, 313, 156, 78.1

Toxicity was observed at the highest or at the two highest dose levels with TA1535 in the absence of S9 metabolism and with TA1537 and TA100 both in the absence and presence of S9 metabolism. Dose-related precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at 5000 and 2500 µg/plate.

As no increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the following dose levels:

Tester strain S9 Dose level (µg/plate)
WP2 uvrA, TA98 ± 5000, 2500, 1250, 625, 313
TA1535 +
TA1535 5000, 2500, 1250, 625, 313, 156
TA1537 +
TA100 ± 2500, 1250, 625, 313, 156, 78.1
TA1537

Toxicity was observed with all tester strains at higher dose levels, both in the absence and presence of S9 metabolism. A more pronounced toxic effect, with toxicity at all or almost all dose levels, was observed with TA1535 and TA1537 in the absence of S9 metabolism and with TA100 both in the absence and presence of S9 metabolism. Dose-related precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at 5000, 2500 and 1250 µg/plate.

In order to assess the mutagenicity of the test item at non cytotoxic concentrations, an additional experiment (Main Assay III) was performed using the pre-incubation method with TA1535 and TA1537, in the absence of S9 metabolism, and with TA100, both in the absence and presence of S9 metabolism. The test item was assayed at the following lower concentrations:

Tester strain S9 Dose level (µg/plate)
TA1535, TA1537 170, 85.0, 42.5, 21.3, 10.6
TA100 ± 85.0, 42.5, 21.3, 10.6, 5.31

Toxicity was observed at the highest or at the two highest dose levels with all tester strains both in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration tested.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assays, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.