Bis(2,3-epoxypropyl) phthalate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,3-epoxypropyl) phthalate

EC Number:

230-566-6

EC Name:

Bis(2,3-epoxypropyl) phthalate

Cas Number:

7195-45-1

Molecular formula:

C14H14O6

IUPAC Name:

bis(oxiran-2-ylmethyl) phthalate

Test material form:

liquid

Details on test material:

As defined in Section 1.2 and 1.4.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, EA 100 did cause a positive mutagenic response with tester strain TA100 and TA1535 in the presence and absence of S9 activation and with tester strain WP2 uvrA in the presence of S9 activation. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.

Executive summary:

The test substance, EA 100, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1000 to 5000 μg per plate with most conditions. Based upon these results, the maximum doses tested in the mutagenicity assay were 5000 μg per plate (for tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and for tester strains TA100 and TA1535 in the presence of S9 activation) and 1000 μg per plate (for tester strains TA100 and TA1535 in the absence of S9 activation).

In the mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 100, 300 and 1000 μg per plate (for tester strains TA100 and TA1535 in the absence of S9 activation) 50.0, 150, 500, 1000, 1500, 3000 and 5000 μg per plate (for tester strains TA100, TA1535 and WP2 uvrA in the presence of S9 activation) and 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate (for tester strains TA98 and TA1537 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation). No precipitate was observed. Toxicity was observed at 5000 μg per plate with some conditions. Positive mutagenic responses were observed (6.7 to 50.3-fold, maximum increase) with tester strains TA100, TA1535 in the presence and absence of S9 activation and WP2 uvrA in the presence of S9 activation.

These results indicate EA 100 was positive for the ability to induce reverse mutations at selected loci of two strains of Salmonella typhimurium (TA100, TA1535) in the presence and absence of an exogenous metabolic activation system and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence of an exogenous metabolic activation system.