Reaction products of 4,4'-(1,3-phenylene-bis(1-methylethylidene))bis-phenol and cyanogen bromide

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:

17th January 2017 to 30th March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

1,3-Bis[2-(4-cyanatophenyl)-2-propyl]benzene

Cas Number:

127667-44-1

Molecular formula:

C26 H24 N2 O2

IUPAC Name:

1,3-Bis[2-(4-cyanatophenyl)-2-propyl]benzene

Constituent 2

Reference substance name:

1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate

Molecular formula:

C78H72N6O6

IUPAC Name:

1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate

Constituent 3

Reference substance name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate

Molecular formula:

C19H19NO

IUPAC Name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate

Constituent 4

Reference substance name:

4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Molecular formula:

C52H50N4O4

IUPAC Name:

4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Constituent 5

Reference substance name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Molecular formula:

C45H45N3O3

IUPAC Name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Constituent 6

Reference substance name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate

Molecular formula:

C38H40N2O2

IUPAC Name:

4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate

Constituent 7

Reference substance name:

2,4,6-tris(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)-1,3,5-triazine

Molecular formula:

C57H57N3O3

IUPAC Name:

2,4,6-tris(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)-1,3,5-triazine

Constituent 8

Reference substance name:

4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Molecular formula:

C104H98N8O8

IUPAC Name:

4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate

Constituent 9

Reference substance name:

Polymeric impurites: reaction product of Bishenol M with cyanogen bromide

Molecular formula:

not available

IUPAC Name:

Polymeric impurites: reaction product of Bishenol M with cyanogen bromide

Details on test material:

Identification: Sodium benzoate
Purity: >99.5%
Physical state/Appearance: white granular solid
Expiry Date: 09 May 2022
Storage Conditions: room temperature over silica gel

Specific details on test material used for the study:

Identification:
4,4’-(1,3-phenylenediisopropylidene) diphenylcyanate
Batch No.:
FAR366085A
Purity:
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials
99.8% (per Certificate of analysis)
Description:
Blackish, yellow-orange, viscous liquid
Storage Conditions:
Room temperature, protected from light
Receipt Date:
13 December 2016

Method

Target gene:

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.

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):

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures;

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

The results of the preliminary toxicity assay conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate in DMSO
The maximum dose of 5000 μg per plate was achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot.

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 350 mg/mL in the solubility test conducted at BioReliance.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Untreated negative 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:

other: yes with metabolic activation

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, 4,4’-(1,3-phenylenediisopropylidene) diphenylcyanate did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, 4,4’-(1,3-phenylenediisopropylidene) diphenylcyanate, 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. Precipitate was observed beginning at 667 or 1000 μg per plate. Toxicity was observed, as reductions in revertant counts, beginning at 333, 3333 or at 5000 μg per plate with most test conditions. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate. Toxicity was observed, as reductions in revertant counts, at 5000 μg per plate with tester strains TA100 and WP2 uvrA in the presence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate 4,4’-(1,3-phenylenediisopropylidene) diphenylcyanate was negative for the 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.