NITTA

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 1991 to 25 January 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Appearance: light brown viscous liquid
- Storage: room temperature

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA1538, TA98 and TA100.

Additional strain / cell type characteristics:

other: histidine-requiring

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced, rat-liver S9.

Test concentrations with justification for top dose:

Concentration range in the preliminary toxicity study: 0, 312.5, 625, 1250, 2500, 5000 µg/plate

Concentration range in the main test (with metabolic activation): 0, 8, 40, 200, 1000, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

Solvent: Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 4-nitro-O-phenylenediamine (5 µg/plate, TA1538 without S9), 2 aminoanthracene (2 µg/plate TA1535 with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Test material and negative controls
0.1 mL of diluted test material or negative control solution was placed in sets of sterile test tubes containing 2.0 mL of molten, trace hstidine supplemented, top agar. These sets comprised of two test tubes for each bacterial tester strain. A 0.1 mL aliquot of one of the bacterial suspensions was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 mix; in the other tube 0.5 mL of pH 7.4 buffer was added. The procedure was repeated in triplicate, for each bacterial strain and for each concentration of test material.

- Positive controls (without activation)
0.1 mL of one of the positive control solutions was added to a test tube containing 2.0 mL of molten, trace hstidine supplemented, top agar at 45 °C. 0.1 mL of the appropriate bacterial suspension and 0.5 mL of pH 7.4 buffer was also added to the test tube. The procedure was repeated in triplicate, for each of the positive controls)

- Positive control (with activation)
0.1 mL of one of the positive control solutions was added to a test tube containing 2.0 mL of molten, trace hstidine supplemented, top agar at 45 °C. 0.1 mL of the appropriate bacterial suspension and 0.5 mL of the S9 mixr was also added to the test tube. The procedure was repeated in triplicate, for each of the positive controls)

The content of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for ca. 48 hours abd tge number of revertant colonies counted.

The complete experiment was repeated using fresh bacterial cultures, test material and control solutions.

Evaluation criteria:

For valid data, the test material was considered to be mutagenic if it induced a dose-related and statistically significant increase in mutation rate in one or more strain of bacteria in the presence/or absence of S9 in both experiments at sub-toxic dose levels.
The test material was considered negative if the number of induced revertants compared to spontaneous revertants was less than twofold at each dose level employed, the intervals of which should be between 2 and 5-fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Preliminary toxicity study
The test material was found to be non-toxic in the strain of Salmonella used (TA100).

- Mutation study
The overnight culture of each strain was found to be in the required range and the spontaneous reversion rate for each was found to be within the expected range.
No toxicity was exhibited to any of the strains of Salmonella tested.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level either with or without metabolic activation.
All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate.

Applicant's summary and conclusion

Conclusions:

The test material was found to be non-mutagenic under the conditions of this test.

Executive summary:

The mutagenicity of the test material was evaluated in a bacterial reverse mutation (Ames) assay which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.14.

During the study triplicate cultures of Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were tested with and without the addition of a mammalian (rat liver) metabolic activation system (S9) in two separate experiments.

No toxicity was exhibited to any of the strains of Salmonella tested. No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level either with or without metabolic activation. All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate.

The test material was therefore found to be non-mutagenic under the conditions of this test.