4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

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:

From 10 March, 2004 to 26 March, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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

Reference substance name:

4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

EC Number:

500-240-0

EC Name:

4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

Cas Number:

68958-77-0

Molecular formula:

UVCB, major component represented by di-functionalised BADGE (HPA-SA-BADGE-SA-HPA): C41H52O16 Other constituents present at >10%: Mono-functionalised BADGE (BADGE-SA-HPA): C35H44O14 Dimers (HPA-SA-BADGE-SA-BADGE-SA-HPA): C66H82O24

IUPAC Name:

2-Propenoic acid, monoester with 1,2-propanediol, polymer with 2-(chloromethyl)oxirane, dihydro-2,5-furandione and 4,4'-(1-methylethylidene)bis[phenol]

Test material form:

liquid

Method

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor-1254 induced rat liver S9-mix).

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Mutation assay: 33, 100, 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: sodium azide (5 µg/plate in saline for TA 1535)

Remarks:

Without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine (60 µg/plate in water for TA1537)

Remarks:

Without metabolic activation

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: daunomycin (4 µg/plate in saline for TA 98)

Remarks:

Without metabolic activation

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (650 µg/plate in DMSO TA100)

Remarks:

Without metabolic activation

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide (10 µg/plate in DMSO for WP2uvrA)

Remarks:

Without metabolic activation

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar

DURATION:
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

COLONY COUNTING: The revertant colonies (histidine independent C.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test substance on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test: The test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100,333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
-Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.
Toxicity: A reduction in the number of revertants equal to the minimal value of the historical control data range is not considered biologically relevant and, therefore, will not be considered cytotoxic. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutation assay: Based on the results of the dose range finding test, the test substance was tested up to concentrations of 3330 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
-Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation on the plates was observed at the start and end of the incubation period at the concentration of 3330 µg/plate.
Toxicity: In both mutation assays, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Number of revertants: In the first mutation experiment an exceedingly low (3 revertants/plate vs historical mean of 7 revertants/plate) solvent control value resulted in a non-dose-related increase in responses observed at all test levels in tester strain TA1537 without metabolic activation. However, no similar 2-fold increase or any dose-related increases in revertants/plate were observed in TA1537 without metabolic activation in an independently repeated experiment

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Refer to attached document under 'Attached background material' for details on results.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance '4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride' according to OECD Guideline 471 and EU Method B.13/14 (Ames test), in compliance with GLP. Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and WP2uvrA of Escherichia coli were exposed to the test substance. The test was performed in two independent experiments in the presence and absence of Aroclor-1254 induced rat liver S9-mix. In the dose range finding test, the substance was tested up to concentrations of 5,000 µg/plate in the strains TA100 and WP2uvrA. The test substance precipitated as of 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at 33 to 3,330 µg/plate in the absence and presence of 5% (v/v) S9-mix in TA1535, TA1537 and TA98. In the second mutation assay, it was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at 3,330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in WP2uvrA both in the absence and presence of S9-metabolic activation, as confirmed in an independently repeated experiment. In this study, the negative and strain-specific control values were within laboratory historical control data ranges, indicating that the conditions were adequate and the metabolic activation system functioned properly. Based on the results, it is concluded that the test substance is not mutagenic in theSalmonella typhimurium and the Escherichia coli reverse mutation assays (Verspeek, 2004).