Reaction products of 2-methylimidazole and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the O.E.C.D. test guideline 471 with GLP compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

Reaction products of 2-methylimidazole and 2,2'-[(1- methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

701-436-0

Molecular formula:

(C15H16O2.C4H6N2.C3H5ClO)x

IUPAC Name:

Reaction products of 2-methylimidazole and 2,2'-[(1- methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Constituent 2

Reference substance name:

EC Substance Name: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 2-methylimidazole

IUPAC Name:

EC Substance Name: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 2-methylimidazole

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

As per IUCLID5 Sections 1.1. 1.2. 1.4. and 4.1.

Method

Target gene:

Histidine synthesis 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:

Rat liver derived S9 fraction with cofactors.

Test concentrations with justification for top dose:

0.50, 1.5, 5.0, 15, 50 and 150 µg per plate with all Salmonella tester strains in the absence of S9 metabolic activation.
1.5, 5.0, 15, 50, 150 and 500 µg per plate with all Salmonella tester strains in the presence of S9 metabolic activation.
15, 50, 150, 500, 1500 and 5000 µg per plate with tester strain WP2 uvrA with and without S9 fraction metabolic activation.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls for non-S9-activated conditions are tester strain specific.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 aminoanthracene for S9 metabolic activation.

Details on test system and experimental conditions:

The Salmonella tester strains were from Dr. Bruce Ames’ Master cultures and the E. coli tester strain was from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Overnight cultures were prepared by inoculating into ~30 to 50 mL of nutrient broth 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 approximately 125 to 175 rpm at 37±2°C approximately 12 to 14 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.

Aroclor 1254-induced rat liver S9 fraction was used as the metabolic activation system. The S9 fraction was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 fraction was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 fraction was stored at 60°C or colder until used. The S9fraction mix was prepared immediately before its use and contained 10% S9 fraction, 5 mM glucose 6 phosphate, 4 mM ß nicotinamide adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4.

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L histidine, D biotin and L tryptophan solution to a final concentration of 50 µM each. Bottom agar was Vogel Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

The bacterial test system was exposed to the test substance via the preincubation methodology described by Yahagi et al. (1977). One half (0.5) milliliter of S9 fraction or posphate buffer mix, 100 µL of tester strain (cells seeded) and 50 µL of DMSO or test substance dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2 C. Colony counts were acquired by a Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments).

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance . Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0 times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value. A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No positive mutagenic responses were observed with any of the tester strains in the presence or absence of S9 fraction metabolic activation when tested up to cytotoxic dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce gene-mutation in any of the bacterial tester strains under the conditions of the study.

Executive summary:

The test substance, Bisphenol A, epichlorohydrin polymer, 2-methylimidazole condensate, was evaluated for gene-mutation potential in an O.E.C.D. test guideline 471 bacterial assay. The test substance did not induce gene-mutation in any of the bacterial tester strains under the conditions of the study.