Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Genetic toxicity: in vitro

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:

2008/06/24 to 2008/07/24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD 471 guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

EC Number:

940-875-4

Molecular formula:

C3H8O3 - C99H182O20

IUPAC Name:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Test material form:

other: viscous amber liquid

Details on test material:

- Physical state: amber liquid or dark orange extremely viscous liquid
- Analytical purity: >99%
- Storage condition of test material: room temprature in the dark without dessicant

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced S9

Test concentrations with justification for top dose:

0, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 ug/plate in the initial test
0, 50, 150, 500, 1500 and 5000 ug/plate in the confirmatory test

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

1000 ug/plate for E.coli

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

1.0 ug/plate for TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1.0 ug/plate for TA100 and TA1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 1 ug/plate for Salmonella, 10 ug/plate for E.coli

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium):

NUMBER OF REPLICATIONS: 3 plates per dose level

NUMBER OF CELLS EVALUATED: at least 10E9 per mL

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of the growth of the background lawn

Evaluation criteria:

For a test article to induce a positive response in the assay, it must cause a dose-related increase in the mean number of revertants per plate in at least one tester strain. The mean number of revertants per plate at the peak of the dose response should be equal to or greater than 2.0 times the mean vehicle control values for tester strains TA98, TAlOO and WP2 uvrA (pKMlO1) and equal to or greater than 3.0 times the mean vehicle control values for tester strains TA1535 and TA1537.

Statistics:

None used

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:

not examined

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:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Control data were within the range of the historical dataset.

Dose formulation analysis confirmed the concentrations of representative concentrations of the test item formulation series.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The summary tables of the results of the two experiments are given in the attachment.

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test, at up to the maximum recommended dose level.

Executive summary:

The purpose of this study was to evaluate the mutagenic potential of GTS26437 by the Bacterial Mutation Test (GLP Ames). The study was conducted at BioReliance, Rockville, MD according to Good Laboratory Practice regulations with Emily W. Dakoulas, B.S., serving as the Study Director. The Study Director signed the protocol on 24 June 2008. The experimental period began on 26 June 2008 and ended on 24 July 2008.

The test article, GTS26437, was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence ofAroclor-induced rat liver S9. The assay was performed in two phases, using both the plate incorporation and the preincubation methods. The first phase, the initial mutagenicity assay via the preincubation method, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay via the plate incorporation method, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide was selected as the solvent of choice based on the Sponsor’s request, solubility of the test article and compatibility with the target cells. Per the Sponsor, the solution was to be sonicated at a minimum of 20 minutes prior to use. In the solubility test, the test article formed a soluble and clear solution in dimethyl sulfoxide at approximately 400 mg/mL with sonication at approximately 25°C for 70 minutes.

In the initial mutagenicity assay, using the preincubation method, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. In the initial mutagenicity assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 ug per plate. No positive mutagenic responses were observed. Neither precipitate nor toxicity was observed. Non-dose responsive increases were observed with tester strain TA98 (1.9-fold) in the presence of S9 activation and with tester strain TA1537 (2.0-fold) in the absence of S9 activation. These were not considered indicative of mutagenic activity the responses were a result of the vehicle control value being on the low end of the acceptable range for TA98 and a single elevated plate count for TA1537. Based on the findings of the initial mutagenicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 ug per plate. In the confirmatory mutagenicity assay, using the plate incorporation method, no positive mutagenic responses were observed with any of the tester strains in the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 ug per plate. Neither precipitate nor toxicity was observed.

Under the conditions of this study, test article GTS26437 was concluded to be negative with Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 wrA (pKM101) in the presence and absence of Aroclor-induced rat liver S9.