Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 26 July 1995 and 1 September 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS harmonised guidelines

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: US EPA (TSCA) guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Method

Target gene:

Not required

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Non-mammalian study

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Range-finding test
Salmonella strains: 0, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain: 0, 50, 150, 500, 1500, 5000 µg/plate

Experiment 2: Main test
Salmonella strains : A0, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification: Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2, 3, 5 ug/plate respectively for WP2uvrA, TA100, TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 ug/plate for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 ug/plate for TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 & TA1537, 10 µg/plate for WP2uvrA , 0.5 µg/plate for TA98

Details on test system and experimental conditions:

Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material
to the tester organisms. A mixture of 0.1 ml of bacterial suspension (TA100 or WP2uvrA-), 0.1 ml of test solution, 0.5 ml phosphate buffer and 2 ml of
molten, trace histidine/tryptophan supplemented media was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Five doses of the
test material and a vehicle control (acetone) were tested in duplicate. After approximately 48 hours incubation at 37°C the plates were scored for
revertant colonies and examined for a thinning of the background lawn.

Mutation Study - Experiment 1 (Range-finding Study):
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
- 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/tryptophan 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 pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, 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 or 4NQO) was added to a test tube containing 2.0 ml of molten, trace histidinel tryptophan 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 an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
- The plates were incubated at 37'C for approximately 48 hours and the number of revertant colonies counted.

Mutation Study - Experiment 2 (Main Study): 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:

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 (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at subtoxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold 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 pg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Results and discussion

Test results

Test results 1

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No toxicity to the bacterial background lawn was exhibited to any of the strains ofbacteria used, although small and inconsistent decreases in revertant colony frequency were observed.

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.

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.

Applicant's summary and conclusion

Conclusions:

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 471, Bacterial Reverse Mutation Assay. The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with 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). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. Decreases in revertant colony frequency were observed in several of the tester strains, but these responses were not accompanied by a weakening of the bacterial background lawn.

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.