Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 July 2014 and 12 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Isobutyl Phenyl Acetate
Chemical nature: 2-methylpropyl 2-phenylacetate
EC No.: 203-007-9
CAS No.: 102-13-6
Molecular weight: 192.2 g/mol
Appearance/physical description: clear, colourless liquid
Empirical formula: C12H16O2
Batch No: SC00010162
Purity: 100%

Method

Target gene:
Salmonella typhimurium: Histidine
Escherichia coli: Tryptophan
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1 - Range-Finding Test) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Test for Mutagenicity (Experiment 2 - Main Test) – Pre-Incubation Method: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle:
Dimethyl sulphoxide
Controls
Negative controls:
yes
Solvent 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: 2-aminoanthracene
Details on test system and conditions:
Test strains
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1408880 10/18) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test and Control Items Preparation
Test Item
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same nominal concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was made for purity. 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. 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.

Control Items
Vehicle, negative (untreated) and positive controls were used in parallel with the test item.

The vehicle control used was dimethyl sulphoxide.
The positive control items used in the series of plates without S9-mix were as follows:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98

In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non mutagenic in the absence of metabolizing enzymes, were used in the series of plates with S9-mix at the following concentrations:

2-Aminoanthracene (2AA): 1 µg/plate for TA100
2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
Benzo(a)pyrene (BP): 5 µg/plate for TA98

Microsomal Enzyme Fraction
Lot No.s PB/βNF S9 02 March 2014 (Experiment 1) and 01 June 2014(Experiment 2) were used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.


S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Top agar was prepared using 0.6% Bacto agar (lot number 3252380 07/18) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 37160 08/14 and 37191 09/14).

Test Procedure
Test for Mutagenicity (Experiment 1 - Range-Finding Test) – 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, 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 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 °C± 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).

Test for Mutagenicity (Experiment 2 - Main Test) – Pre-Incubation Method
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 (main test) was determined by the results of Experiment 1 (the range-finding test) and was 5 to 5000 µg/plate.
Seven test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C± 3 °C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 °C± 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).
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.     Statistical analysis of data as determined by UKEMS (Mahonet 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.
 
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first experiment, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 and WP2uvrA dosed in the presence of S9-mix), initially from 1500 μg/plate. Consequently the same maximum dose level was used in the second mutation test. After employing the pre-incubation modification in Experiment 2, the test item was noted to have exhibited toxicity as weakened bacterial background lawns to all of the tester strains (except WP2uvrA in the presence of S9-mix), initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. A slight, globular precipitate was observed under the dissection microscope at 5000 μg/plate in both the presence and absence of S9-mix but was not visible by eye.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 5 μg/plate in the second mutation test. This increase was
considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 5 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Isobutyl Phenyl Acetate was considered to be non-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 typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia colistrain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre‑incubation methods at up to 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 the range-finding test (Experiment 1) was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and was 5 to 5000 µg/plate.

 

Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

 

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. In the first experiment, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 and WP2uvrA dosed in the presence of S9-mix), initially from 1500 μg/plate. Consequently the same maximum dose level was used in the second mutation test. After employing the pre-incubation modification in Experiment 2, the test item was noted to have exhibited toxicity as weakened bacterial background lawns to all of the tester strains (except WP2uvrA in the presence of S9-mix), initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. A slight, globular precipitate was observed under the dissection microscope at 5000 μg/plate in both the presence and absence of S9-mix but was not visible by eye.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 5 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 5 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.4 times the concurrent vehicle control.

 

Conclusion

Isobutyl Phenyl Acetate was considered to be non-mutagenic under the conditions of this test.