Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2 yl)phenoxy]methyl}oxirane and 2RS)-2-({4-[2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane

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:

15t March 2018 to 30th April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene -2- yl)phenoxy] methyl} oxirane

Molecular formula:

C20 H18 Cl2 O4

IUPAC Name:

meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene -2- yl)phenoxy] methyl} oxirane

Test material form:

solid

Details on test material:

Batch GRM193K01
Manufactured 19.07.2019
exp date : 03.09.2021
Density 1.32 g/cm3 at 23°C

Specific details on test material used for the study:

REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE

Description:
Whitish yellow solid
Storage Conditions:
Room temperature, protected from light
Receipt Date:
09 February 2018

Purity:
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

Method

Target gene:

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.

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

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.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3880, Exp. Date: 31 Oct 2019) was purchased commercially from MolTox (Boone, NC).

Concentrations in the preparations can be found in Table 1.

Test concentrations with justification for top dose:

In the initial toxicity-mutation screen, a maximum dose of 5000 μg per plate was achieved using a concentration of 100mg/mL and a 50.0 μL plating aliquot. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. No positive mutagenic responses were observed.

Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive 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.

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

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.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls.

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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed.
BioReliance Study No. AF23LZ.503.BTL 17
Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation.

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, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE did cause a positive mutagenic response with tester strain TA100 and TA1535 in the presence or absence of S9 activation and WP2 uvrA in the absence of S9 activation.

Executive summary:

The test substance, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE, 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 initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. The tester strain WP2 uvrA in the presence of S9 activation was not evaluated as the top three dose levels were plated without tester strains due to technician error. This condition was repeated. Precipitate was observed beginning at 1500 μg per plate with all conditions (except WP2 uvrA in the presence of S9 activation). No toxicity was observed (except WP2 uvrA in the presence of S9 activation). Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation. Based upon these results, the maximum dose tested in the retest of initial toxicity-mutation assay and confirmatory mutagenicity assay was 5000 μg per plate.

In the retest of initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate with WP2 uvrA in the presence of S9 activation. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. No positive mutagenic responses were observed.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation.

These results indicate REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE was positive 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.