Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-{2-[(2-ethylhexyl)oxy]ethoxy}ethyl prop-2-enoate
Cas Number:
117646-83-0
Molecular formula:
C15H28O4
IUPAC Name:
2-{2-[(2-ethylhexyl)oxy]ethoxy}ethyl prop-2-enoate
Test material form:
liquid
Details on test material:
Test substance name: MI RAMER M1086
Appearance : Clear liquid
Manufacturing date: 2012-03-29
Supplier:Miwon Specialty Chemical Co., Ltd.
Receipt day: 2012-05-23
Deli very amount: 399.223 g(Gross)
Cas No.:117646-83-0
Lot No.:120329JY1
Purit y: 95.55 %
Expiration date: 2013-03-28 (Manufactured after 1 year)
Storage condition: Room temperature [(15 ~ 25)°C
Specific details on test material used for the study:
Name : MIRAMER M1086
Supplier : Miwon Specialty Chemical Co., Ltd.
Manufactured date : 2012-03-29
Delivery date : 2012-05-23
Delivery amount : 399.223 g (Gross)
Appearance : Clear liquid
Lot No. : 120329JY1
Cas No. : 117646-83-0
Purity : 99.5 %
Molecular weight : 272
Storage condition : Room temperature (15-25°C)
Expiration date : 2013-03-28

Method

Target gene:
Base pair substitution type : Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2uvrA
Frame-shift type : Salmonella typhimurium TA98 and TA1537
The test strains listed below were chosen according to the OECD guidelines, and they are known to be very sensitive to mutagens and widely used in mutagenicity studies with bacteria. All of the test strains were purchased from Molecular Toxicology Inc. (2011-03 ~ 2011-05) and used after subculture following confirmation of genotypes.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Test concentrations with justification for top dose:
Preparation of test article:
The does range-finding test was performed at concentration levels of 50, 100, 500, 1000 and 5000 μg/plate (the highest concentration). Based on the dose range-finding test, 100 μg/plate was selected as the highest concentration of the main test, and the highest concentration was serially two-fold diluted to make 5 concentration levels.
Preparation of positive controls:
Sodium azide was dissolved in distilled water while other three positive controls were dissolved with DMSO, and then all of positive controls were stored at -20°C. These prepared positive items were thawed immediately before use.
Vehicle / solvent:
The test article was soluble in DMSO, so DMSO was chosen as a vehicle and usedin diluting the test article. The chemicals described in OECD guideline were chosen as positive control article.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Treatment of test article
Treatment of test article was tested by using pre-incubation method. 0.1 mL of test article, 0.5 mL of S9 mix (or sodium phosphate buffer, pH 7.4) and 0.1 mL of bacterial culture were added to each dry heat sterilized test tube. This mixture was shaken for 20 minutes at 37°C using shaking incubator (120 rpm). Next, 2 mL of top agar was added to each test tude, and then the mixture in the test tubes was poured onto minimal glucose agar plate (one tube per plate). Vehicle control groups were treated with vehicle only, and each of positive control groups was treated with strain-specific positive controls. After the top agar was solidified, the plates were inverted and incubated at 37°C for about 48 hours. Then, the revertant colonise were counted. In order to check sterility in this test, the highest concentration of test article (0.1 mL) and S9 mix (0.5 mL) were mixed individually with 2 mL of top agar and poured onto agar plate in the same manner.

Dose range-finding test
Dose range-finding test was performed with the five test strains at dose levels of 0, 50, 100, 500, 1000 and 5000 μg/plate both in the absence (S9-) and presence (S9+) of S9 mix. Each dose group was assayed in triplicate.

Main test
100 μg/plate was selected as the highest concentration for the main test by considering the result of dose ranging-finding test, and the highest concentration was serially two-fold diluted to make 5 concentration levels (6.25, 12.5, 25, 50 and 100 μ g/plate). Each dose group was assayed in triplicate.

Conformation test was not performed.

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:
no cytotoxicity
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:
no cytotoxicity
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:
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:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Test article, MIRAMER M1086, was evaluated for its potential to induce reverse mutation in four histidine auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one tryptophan auxotroph strain of Escherichia coli (WP2uvrA ).
The test article, MIRAMER M1086, was dissolved and diluted with DMSO. Precipitation or anything unusual to report was not observed during the test article preparation.
The main test was performed at five concentration levels of 6.25, 12.5, 25, 50, 100 μ g/plate with negative and positive controls in the absence and presence of metabolic activation system (S9 mix). There was no contamination from the test article and S9 mix used in the main test. The number of viable cell counted in overnight cell cultures for the five strains used in the test, measured by the serial dilution method, was 2.1 ~ 5.2×109 CFU/mL which was considered to be appropriate.
There was no reproducible increase in the number of revertant colonies compared to its vehicle control at any dose in any of the strains. The positive controls induced a marked increase in the number of revertant colonies as anticipated.
Therefore, it is considered that test article, MIRAMER M1086, showed no evidence of mutagenic potential at the concentration range tested in five test strains under the condition of this test.
Executive summary:

The test article, MIRAMER M1086, was evaluated for its potential to induce reverse mutation in the histidine auxotroph strains of Salmonella typhimurium such as TA100, TA1535, TA98 and TA1537 and in the tryptophan auxotroph strain of Escherichia coli WP2uvr A.

The test article was dissolved and diluted with DMSO. Based on the dose range-finding test which was performed at concentrations of 50, 100, 500, 1000 and 5000 μg/plate (the highest concentration), the main test was performed with the final concentrations of test article ranged 6.25, 12.5, 25, 50 and 100 μg/plate with negative and positive control in the absence and presence of metabolic activation system (S9

mix).

In this study, there was no significant increase in the number of revertant colonies compared to its vehicle control at any dose in any of the strains. In addition, antibacterial effects such as a decrease in the number of colonies were also not observed in any of the strains.

Therefore, these results of the bacterial reverse mutation assay showed that, under the conditions of this study, MIRAMER M1086 was considered not to induce a reverse bacterial mutation to the strains used in this test.