POLYPERINY

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:

1996-05-15 - 1996-06-07 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1997

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:

solid: particulate/powder

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

50 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5 µg/plate 4-nitro-o-phenylenediamine for the strain TA 1538

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA), 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 0.5 µg/plate for TA1538 and TA98

Remarks:

with S-9 metabolic system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Preparation of Test and Control Materials:
The test material was accurately weighed and approximate half-log suspensions in dimethyl formamide prepared by action on an autovortex and sonication for 15 minutes on the day of each experiment. The dosing solutions were also vortexed throughout the course of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined.
Vehicle and positive controls were used in parallel with the test material. In addition the material, 2-Aminoanthracene (2AA), which is non-mutagenic in the absence of metabolising enzymes was used in the S9 series of plates

DURATION
- Expression time (cells in growth medium): All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter

NUMBER OF REPLICATIONS: 3

Mutation Study - Experiment 1
Five concentrations of the test material were assayed against each tester strain.
Test Material and Vehicle Controls:
Known aliquots (0.1 ml) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar at 45 °C, 0.1 ml of the appropriately diluted test material or vehicle control and either 0.5 ml of the S9 liver microsome mix or 0.5 ml of phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated for each bacterial strain and for each concentration of test material with and without S9-mix.
Positive Controls
Without Activation: A known aliquot (0.1 ml) of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of pH 7.4 buffer was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated for each tester strain.
With Activation: A known aliquot (0.1 ml) of 2AA solution was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of S9-mix was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated for each tester strain.
Mutation Study - Experiment 2
The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Evaluation criteria:

Interpretation of Results
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS (5) and normally Dunnett's method of linear regression is used to evaluate the result. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test results

Test results 1

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strain of Salmonella used (TA100).

Applicant's summary and conclusion

Conclusions:

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.

Executive summary:

The mutagenic potential of the test substance was investigated according to the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC.

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. No toxicity was exhibited to any of the strains of Salmonella used. A precipitate was observed at and above 1500 µg/plate, this did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level with or without metabolic activation.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

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