Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Specific details on test material used for the study:
Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor.
Identification: EP506
Physical state/Appearance: Pale yellow solid
Chemical Name: Phenol, 4,4'-(1-methylethylidene)bis[2,6-dibromo-, polymer with 2-(chloromethyl)oxirane and 4,4'-(1-methylethylidene)bis[phenol], Ph ethers
CAS Number: 1045809-53-7
Batch Number: DG7H30082A
Purity: 100% (UVCB)
Expiry Date: 23 August 2020
Storage Conditions: Room temperature in the dark
No correction for purity was required.

Method

Target gene:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitution
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Experimental Design and Study Conduct

Test Item Preparation and Analysis
The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

The test item was accurately weighed and, on the day of the experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction for purity was required. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 µg/plate because of a test item film.

Test for Mutagenicity: Experiment 2
The second experiment was not performed because the OECD 471 test guideline permits non-repetition of the experiment when a clear, positive response is obtained in the first experiment. The results of Experiment 1 confirmed that a second experiment was not required.
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:

TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

There should be a minimum of four non-toxic test item dose levels.

There should be no evidence of excessive contamination.

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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item induced statistically significant and dose-related increases in the frequency of TA100 and TA1535 revertant colonies in excess of twofold the concurrent vehicle controls initially from 150 µg/plate in the absence of S9-mix (TA100) and 500 µg/plate in the presence of S9-mix (TA1535). The majority of statistically significant increases observed in both the absence of presence of S9-mix for TA100 and TA1535 were large and in excess of the in-house historical control range for the relevant bacterial strains with a maximum increase of 3.5 and 4.5 times over the concurrent vehicle control (absence and presence of S9-mix) noted for TA1535 at 5000 µg/plate. Any excursions outside the maxima ranges, particularly when a dose-response relationship is apparent, must be considered to be evidence of a biological response. A weaker statistically significant response was also noted to WP2uvrA dosed in the absence of S9-mix at 5000 µg/plate, however these counts were within the in-house untreated/vehicle historical control profile for the strain.

Applicant's summary and conclusion

Conclusions:
The test item, EP506 was considered to be mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008

and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).  The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate.

The OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment, therefore, with the Sponsor’s approval, testing was suspended at the end of Experiment 1.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases

in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.  There was no visible reduction in the growth of

the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the mutation test (plate incorporation method).  

An opaque test item film was observed from 1500 µg/plate in both the presence and absence of S9-mix.  This observation did not prevent the scoring of revertant colonies.

The test item induced statistically significant and dose-related increases in the frequency of TA100 and TA1535 revertant colonies in excess of twofold the concurrent vehicle controls initially from 150 µg/plate in the absence of S9-mix (TA100) and 500 µg/plate in the presence of S9-mix (TA1535).  The majority of statistically significant increases observed in both the absence of presence of S9-mix for TA100 and TA1535 were large and in excess of the in-house historical control range for the relevant bacterial strains with a maximum increase of 3.5 and 4.5 times over the concurrent vehicle control (absence and presence of S9-mix) noted for TA1535 at 5000 µg/plate.  Any excursions outside the maxima ranges, particularly when a dose-response relationship is apparent, must be considered to be evidence of a biological response.  A weaker statistically significant response was also noted to WP2uvrA dosed in the absence of S9-mix at 5000 µg/plate, however these counts were within the in-house untreated/vehicle historical control profile for the strain.

Conclusion

The test item, EP506 was considered to be mutagenic under the conditions of this test.