Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 12, 2017 to January 12, 2018
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 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
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Version / remarks:
24 November 2000
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:
- Appearance: Clear liquid
- Moisture content: 0.02 %
- Stabilisation: polymerisation inhibitor 253 ppm MEHQ

Method

Target gene:
Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli
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:
Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1 (Ames plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (Pre-incubation method): 15, 50, 150, 500, 1500, 5000 µg/plate
- Salmonella typhimurium strain TA100 required a repeat second experiment analysis due to elevated untreated/ vehicle control counts and, as toxicity was noted, the dose range was amended:
TA100 (presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Controls
Untreated negative controls:
no
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:
Test Item Preparation and Analysis

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. No correction was required for purity. 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 together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. 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 ± 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 – Pre-Incubation Method

As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

- Dose Selection
The dose range initially used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 µg/plate. Six test item dose levels per bacterial strain were originally selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation. Salmonella strain TA100 required repeat second experiment analysis due to elevated untreated/ vehicle control counts and, as toxicity was noted in the second mutation test, the dose range for this particular tester strain was amended as follows:

TA100 (presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate

- 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 or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace 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 (see 3.3.3.2) 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 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

- 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).
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:

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.

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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A test item precipitate (greasy in appearance) was noted in both experiments at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in Experiment 1 (presence of S9-mix) at 50 µg/plate. However, this response was within the in-house historical vehicle/untreated control values for the bacterial strain and was, therefore, considered of no biological relevance. Subsequently, there were no toxicologically meaningful 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (S9-mix) in Experiment 2 (pre-incubation method).
Remarks on result:
other: The toxic limit of the test item (1500 µg/plate) was employed in the absence of S9 for this strain only

Applicant's summary and conclusion

Conclusions:
2-Butoxyethyl methacrylate was considered to be non-mutagenic under the conditions of this bacterial reverse mutation assay (Ames Test) in Salmonella typhimurium and Escherichia coli.
Executive summary:

An in vitro study was undertaken to determine the capacity of the 2-butoxyethyl methacrylate to induce reverse gene mutation at the histidine or tryptophan locus in five bacterial strains: Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA). The experiment was performed without deviation according to guidelines published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guideline 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.

An initial experiment (Experiment 1) was performed using the Ames plate incorporation technique at pre-determined dose levels of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (n = 3) with and without metabolic activation, which consisted of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). A subsequent test (Experiment 2) was performed using the pre-incubation method based on the outcome of Experiment 1, with dose levels amended to 15, 50, 150, 500, 1500, 5000 µg/plate in order to achieve four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology. Strain TA100 required repeat analysis as part of Experiment 2 due to elevated vehicle control counts, and as toxicity was noted in the second mutation test, the dose range for this strain was amended to 15 - 5000 µg/plate (presence of S9-mix) and 1.5 to 1500 µg/plate (absence of S9-mix). The vehicle control was untreated and contained dimethyl sulphoxide. Five positive control substances were prepared in addition. 

According to the results of the vehicle, positive controls and negative controls, the sensitivity of the assay and the efficacy of the S9-mix were validated. No toxicologically meaningful 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 (S9-mix) in Experiment 1 and 2. As 2-butoxyethyl methacrylate did not have the capacity to significantly induce gene mutation in bacteria under the conditions of this test, it can be regarded as non-mutagenic.