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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
Study period:
From August 11, 1999 to August 19, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471, EPA OPPTS 870.5100 and EU Method B.13/14 in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reaktiv - Orange F97-0318

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
First plate incorporation test
a: without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate
b: with metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Second plate incorporation test:
a: without metabolic activation:
500, 1,600, 2,000, 3,000, 4,000 and 5,000 µg/plate (TA 1535)
b: with metabolic activation:
500, 1,600, 2,000, 3,000, 4,000 and 5,000 µg/plate (TA 1535)
Vehicle / solvent:
Deionized water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation for strain TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation for strain WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (i.e., S9-mix from rat liver) for all strains
Details on test system and experimental conditions:
Assay procedure

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 5,000 µg/plate. Further dilutions of 1,600, 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 (i.e., 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 (i.e., 2.5 mL, 0.5 mM). The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 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 substance solution (i.e., dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 min at approximately 30ºC.

After mixing, and pre-incubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (i.e., 1.5% (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 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 assay is considered valid if the following criteria are met:
- 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 test substance is classified as mutagenic if it has either of the following effects:
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 substance 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
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and toxicity

-The test substance was dissolved in deionized water 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 1,600, 500, 160 and 50 µg/plate were used in the first plate incorporation test.

-The test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.

-Because of mutagenic effects with the strain TA 1535 a second plate incorporation test was performed with this strain at with dose levels from 500 to 5,000 µg/mL and the concentrations of 2,000, 3,000 and 4,000 µg/plate were included in the treatment series.

-In both plate incorporation tests toxicity was not observed with and without metabolic activation.

Mutagenicity

-In both independent mutation tests, test substance was tested for mutagenicity with the same stated concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.

-In the absence and in the presence of the metabolic activation system the test substance induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strain TA 1535.

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

Any other information on results incl. tables

Sterility checks and control plates

 

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance and S9-mix sterility check plates. Control plates (i.e., background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation (with the bacterial strain TA 1535)
positive without metabolic activation (with the bacterial strain TA 1535)

Under the study conditions, the test substance was found to be mutagenic in the bacterial reverse mutation assay.
Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100 and EU Method B.14, in compliance with GLP.

 

One independent mutagenicity study was conducted, as the plate incorporation method. The study was performed in the absence and the presence of a metabolizing system derived from a rat liver homogenate (i.e., S9 mix). The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate. Additionally the plate incorporation test was repeated with the strain TA 1535 with and without S9-mix at six concentrations in the range of 500 - 5,000 µg/plate.

 

In both plate incorporation tests, the substance induced a dose-dependent increase in the number of revertant colonies with theSalmonella strain TA 1535 in the absence and presence of the S9 mix. Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. No toxicity was observed in any of the assays.

 

Under the study conditions, the test substance was found to be mutagenic in the bacterial reverse mutation assay.