Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06/Feb/2019 - 04/Mar/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries,
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register.
Version / remarks:
Adopted 2012; 77:33748-33749)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
4,4’-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid
EC Number:
948-054-2
Molecular formula:
not applicable - the substance is an UVCB
IUPAC Name:
4,4’-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid
Test material form:
liquid

Method

Target gene:
Histidine metabolism for Salmonella strains.
Tryptophan metabolism for Escherichia strain.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: HSD: CD Sprague Dawley
- method of preparation of S9 mix: Oral, Phenobarbital / Beta-Naphtha flavone at 80/100mg/Kg
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9 fraction in S9 mix. 0.5 mL used.
- quality controls of S9: sterility and efficacy, certificate provided.
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: Test item immiscible in sterile distilled water, but fully miscible in DMSO.

- Justification for percentage of solvent in the final culture medium: To be in line with in-house historical control profiles.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: Identity: 2-Aminoanthracene (2AA) CAS No.: 613-13-8 Batch number: STBB1901M9 Purity: 97.5% Expiry date: 08 October 2019 Solvent: DMSO Concentration: 1 ug/plate for TA100 2 ug/plate for TA1535 and TA1537 10 ug/plate for WP2uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 10 hours.
- Harvest time after the end of treatment: after 48-72 hours .

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48-72 hours.
- Method used: agar.
- Method to enumerate numbers of viable and mutants cells: automated colony counting system.
Rationale for test conditions:
In accordance with the OECD Testing Guideline 471.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
statistically significant increases in the frequency of revertant colonies
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
other: non statistically significant increases in the frequency of revertant colonies
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
other: non statistically significant increases in the frequency of revertant colonies
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
other: non statistically significant increases in the frequency of revertant colonies
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
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
True negative controls validity:
not applicable
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL used.
- Precipitation and time of the determination: Cream coloured test item film discovered at time of counting.

RANGE-FINDING/SCREENING STUDIES (if applicable): Experiment one informed ranged used in Experiment 2.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Available in attached background documents.

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: * = p < 0.05.

Ames test:
- Signs of toxicity : None mentioned.
- Individual plate counts : Available in attached background documents.
- Mean number of revertant colonies per plate and standard deviation : Available in attached background documents.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Available in attached background documents.
- Negative (solvent/vehicle) historical control data: Available in attached background documents.

Any other information on results incl. tables

Full results tables can be found in the attached background documents.

Only TA100 results in the absence of metabolic activation are described as statistically significant.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test 4,4'-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid was considered to be mutagenic.
Executive summary:

A GLP-compliant bacterial reverse mutation assay was performed on 4,4'-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid in accordance with the OECD Testing Guideline 471. The purpose of the assay was to evaluate 4,4'-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

The results were that it induced a dose related, statistically significant and reproducible increase in the frequency of TA100 revertant colonies. This happened only at the upper dose levels, in the absence of metabolic activation. Smaller, non-statistically significant increases in revertant colonies were observed in some strains of bacteria the absence and/or presence of metabolic activation.

Because of this, it was concluded that 4,4'-isopropylidenediphenol, reaction products with 1-chloro-2,3-epoxypropane, mono, di and triesters with acrylic acid was mutagenic under the conditions of the test.