Registration Dossier

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
Study period:
March to April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD method without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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

Constituent 1
Chemical structure
Reference substance name:
4,4'-(2-ethylhexane-1,1-diyl)diphenol
EC Number:
680-046-1
Cas Number:
74462-02-5
Molecular formula:
C20H26O2
IUPAC Name:
4,4'-(2-ethylhexane-1,1-diyl)diphenol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: 4,4’-(2-Ethylhexylidene)bisphenol
Other name: BisP-IOTD
CAS nunber: 74462-02-5
Lot number: 120601
Supplier and date of receipt: Honshu Chemical Industry Co., Ltd., February 25, 2013
Amount of supplied, used: 10 g, approximately 0.44 g
Purity: 99.6%
Molecular weight: 298.4
Melting point: 88.9°C
Description at ordinary temperature: white powder
Stability: stable at room temperature
Solubility: slightly soluble in water, dissolves in acetone at 100 w/v% , insoluble in water, dissolves in dimethylsulfoxide at 5 wt% or more and at 10 wt% or more in acetone
Stability in solvent: stable in water, dimethylsulfoxide and acetone
Storage conditions: sealed, room temperature (1 to 30 °C)
Place of storage: room temperature cabinet in the dispensary of the main research building (allowable range of temperature, 1 to 30 °C)
Actual storage temperature: 18 to 21 °C
Storage period: February 25, 2013 to March 25, 2013

Method

Target gene:
The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
dose finding study: 15.8, 50.0, 158, 500, 1580, 5000 µg/plate (with and without S9)
main study: 5.0, 10.0, 20.0, 40.0, 80.0 and 160 µg/plate (without S9) and 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate (with S9)
confirmatory study: 3.75, 7.50, 15.0, 30.0, 60.0, 120 µg/plate (without S9) and 11.3, 22.5, 45.0, 90.0, 180 and 360 µg/plate (with S9)
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
with and without metabolic activation
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene
Details on test system and experimental conditions:
The studies were performed in the presence and absence of metabolic activation in each strain. The negative and positive controls were also included. Minimum glucose agar plates were prepared in duplicate per dose for the test substance, negative and positive control groups in the dose-nding study and in triplicate in the main and conrmatory studies. The doses of positive control substances, which have been generally used in
bacterial reverse mutation tests, are shown below.
TA100 without metabolic activation: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide 0.01 µg/plate
TA100 with metabolic activation: 2-aminoanthracene 1.0 µg/plate
TA1535 without metabolic activation: Sodium azide 0.5 µg/plate
TA1535 with metabolic activation 2-aminoanthracene: 2.0 µg/plate
WP2 uvrA without metabolic activation 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate
WP2 uvrA with metabolic activation 2-aminoanthracene: 10.0 µg/plate
TA98 without metabolic activation 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide: 0.1 µg/plate
TA98 with metabolic activation 2-aminoanthracene: 0.5 µg/plate
TA1537 without metabolic activation 9-aminoacridine: 80.0 µg/plate
TA1537 with metabolic activation 2-aminoanthracene: 2.0 µg/plate

Preparation of S9 mix
The frozen S9 at -80 °C was thawed and was mixed with Co-factor solution prior to use in the ratio of 1:9. The composition in 1 mL of S9 mix is shown below. The prepared S9 mix was refrigerated in an ice bath.
S9 0.1 mL
MgCl2 8 µmol/mL
KCl 33 µmol/mL
Glucose-6-phosphate 5 µmol/mL
NADPH 4 µmol/mL
NADH 4 µmol/mL
Sodium phosphate buffer, pH 7.4 100 µmol/mL
Evaluation criteria:
The test results were judged positive when biologically meaningful increase in the number of revertant colonies, such as dose-related increase with reproducibility were observed.
Statistics:
The number of revertant colonies per plate, the mean values and standard deviation per dose of the test substance, and the negative control were tabulated for each strain. The number of revertant colonies per plate and the mean values were tabulated for each strain as for the positive control. Dose-response curves of each strain were drawn for the test substance group.
Two statistical analyses of Dunnett’s multiple comparison method (one-side test) and linear regression method were used in this test. The number of revertant colonies of each bacterial strain at each dose in the main study and confirmatory study was compared with that of the negative control in both the presence and absence of metabolic activation, and statistically significant difference in the number of revertant colonies between those two groups was analyzed first by the multiple comparison method (p<0.05). The dose-reactivity was analyzed by linear regression method (p<0.05) when the statistically significant difference was detected by the multiple comparison method.

Results and discussion

Test resultsopen allclose all
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
Remarks:
at 158 µg/ml (without S9) and 500 µg/ml (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 158 µg/ml (without S9) and 500 µg/ml (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose-finding Study
The test substance showed growth-inhibition at the dose of 158 µg/plate or more in the absence of metabolic activation and at the dose of 500 µg/plate or more in the presence of metabolic activation. On the other hand, increase in the number of revertant colonies compared with the negative control was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Precipitation of the test substance was observed at the dose of 5000 µg/plate in the absence of metabolic activation and at the dose of 1580 1.1g/plate or more in the presence of metabolic activation.
Main Study
The test substance showed growth-inhibition at the dose of 80.0 µg/plate or more in the absence of metabolic activation and at the dose of250 µg/plate or more in the presence of metabolic activation. On the other hand, increase in the number of revertant colonies compared with the negative control was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Precipitation of the test substance was not observed at any dose levels regardless of the presence or absence of metabolic activation.
Confirmatory Study
The test substance showed growth-inhibition at the dose of 60.0 µg/plate or more in the absence of metabolic activation and at the dose of 360 µg/plate in the presence of metabolic activation. On the other hand, increase in the number of revertant colonies compared with the negative control was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Precipitation of the test substance was not observed at any dose levels regardless of the presence or absence of metabolic activation.

Any other information on results incl. tables

Reverse mutation test of 4,4’-(2-Ethylhexylidene)bisphenol was performed using five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA, in the presence and absence of metabolic activation with a pre-incubation method at 37 °C for 20 minutes.

Statistically significant difference in the number of revertant colonies between the test substance group and the negative control was not observed at any dose levels in any bacterial strains regardless of the presence or absence of metabolic activation. Reproducibility was observed between the results from the main study and confirmatory study. These results revealed that no biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was observed. The number of revertant colonies in the positive control was twice or more than that of the negative control in all bacterial strains in both the presence and absence of metabolic activation. The mean values of colony counts in the negative and positive controls were within the range of background data both in the main study and confirmatory study. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor that affected the reliability of the studies was confirmed.

Based on the above results, the test substance was considered to have no potency of mutagenicity under the conditions of this test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance 4,4'-(2-Ethylhexylidene)bisphenol was found negative (with and without metabolic activation) in this Ames-bacterial reverse mutation assay using salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as E. coli strain WP2 uvrA.
Executive summary:

The inducibility of gene mutation in 4,4'-(2-Ethylhexylidene)bisphenol was evaluated by the reverse mutation test with a pre-incubation method at 37 °C for 20 minutes using five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA. The test was composed of the dose-finding, main and confirmatory studies, and the reproducibility between the results from the main and confirmatory studies was confirmed. All studies were performed in the presence and absence of metabolic activation. As a result, biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. The number of revertant colonies in the positive control was twice or more than that of the negative control in all bacterial strains regardless of the presence or absence of metabolic activation. The mean values of revertant colonies in the negative and positive controls were within the range of calculated reference value from background data in the main and confirmatory studies. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor that affected the reliability of the studies was confirmed.

Based on the above results, the inducibility of gene mutation in the test substance was determined as negative under the test conditions employed.