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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Standard for Investigation by Mutagenicity Studies using Microorganisms Sept. 1, 1988 ML Notification No. 77 Amended on June 2, 1997 ML Notification No. 67 Amended on December 25, 2000 ML Notification No. 120
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
plates of the test item in duplicate, no statistical analysis
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: Colorless clear liquid
- Storage Conditions: Room temperature

Method

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:
S9 mix
Test concentrations with justification for top dose:
Dose finding test
50, 12.5, 3.13, 0.781, 0.195, 0.0488 and 0.0122 mg/ mL.

Main test
12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mg/mL.
Vehicle / solvent:
From the results of the solubility test which was conducted at water and DMSO, DMSO was used as the vehicle in this study since the test article was insoluble at 50 mg/mL in water and dissolved at 50 mg/ mL in DMSO, and there were no observed reactions such as exothermic reaction or generation of gasses. DMSO dehydrated with molecular sieves 4A 1/16 (Wako Pure Chemical Industries, Ltd.: Lot No. MPG7052) was used since there is no information from the sponsor on the stability in water.

Name: DMSO
Manufacturer: Wako Pure Chemical Industries, Ltd.
Lot Number: PDR5321
Specification: JLS Reagent Special grade, not less than 99.0%
Storage Conditions: Room temperature
Storage: The test article preparation room at Tokyo Laboratory
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-FuryI)-3-(5-nitro-2-furyl)acrylamide; 2-Methoxy-6-chloro-9-[3-(2-cholroethyl)aminopropylamino]acridine 2HCl; 2-Aminoanthracene
Details on test system and experimental conditions:
Preparation Methods of Test Solutions
Dose-finding Test
The test article (0.200 mL) was put into a sterilized test tube for preparation, and weighed using an electric balance (electronic balance GR-120, A&D Co., Ltd.). The amount of vehicle in the test solution at the highest concentration of 50 mg/mL was calculated for the weighed value (217.0 mg). Then, 4.140 mL of DMSO, which was calculated by subtracting the volume of the test article (0.200 mL) from the calculated volume, was added to prepare the test solution at 50 mg/mL. This solution was diluted 6 times using a common ratio of 4 to prepare test solutions at a total of 7 concentrations: 50, 12.5, 3.13, 0.781, 0.195, 0.0488 and 0.0122 mg/mL. During the time of preparation, there were no reactions such as exothermic reaction or generation of gasses.
Test solutions were prepared at the time of use under fluorescent lamps with ultraviolet absorbing films.

Main Test
The test article (0.070 mL) was put into a sterilized test tube for preparation, and weighed using an electric balance (electronic balance GR-120, A&D Co., Ltd.). The amount of vehicle in the test solution at the highest concentration of 12.5 mg/mL was calculated for the weighed value (76.2 mg). Then, 6.026 mL of DMSO, which was calculated by subtracting the volume of the test article (0.070 mL) from the calculated volume, was added to prepare the test solution at 12.5 mg/mL. This solution was diluted 7 times using a common ratio of 2 to prepare test solutions at a total of 8 concentrations: 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mg/mL. During the time of preparation, there were no reactions such as exothermic reaction or generation of gasses. Test solutions were prepared at the time of use under fluorescent lamps with ultraviolet-absorbing films.
The test was performed in duplicate.

Strains Used
The following 5 strains were used.
Base-pair substitution type:
S. typhimurium TA 100
S. typhimurium TA 1535
E, coli WP2 uvrA
Frame-shift type:
S. typhimurium TA 98
S. typhimurium TA 1537
S. typhimurium TA strains were obtained fiom the Division of Genetics and Mutagenesis, National Institute of Health Sciences by Gotemba Laboratory, Bozo Research Center Inc. on October 9, 1997. Batches of the strains were separated fiom those stored by Gotemba Laboratory, Bozo Research Center Inc. and transferred to Tokyo Laboratory, Bozo Research Center Inc. on July 21, 2005. E. coli WP2 wrA was obtained fiom the National Institute of
Technology and Evaluation on October 20,2011.

Storage and Thawing of Tester Strains
The frozen-stock cell lines passaged fiom the original tester strains were cultured and 0.7 mL of DMSO (Wako Pure Chemical Industries, Ltd., JIS Reagent Special Grade, Lot No. PDR5321) was added to 8.0 mL of each bacterial solution. These tester strain solutions were placed in sterilized tubes (0.3 mL each), then were rapidly frozen with dry ice-acetone and stored in a deep freezer (SANYO Electric Biomedical Co., Ltd.: MDF-192) set at -70°C or below (measured temperature during the storage period from August 1, 2013 to August 21, 201 3: -87.0 to -78.1°C). They were thawed at room temperature at the time of use and the residue was discarded after culturing.

Characteristics Tests of Tester Strains
At the time of preparation of frozen-stock cell lines, the tester strains were examined for the following characteristics: amino acid requirement, rfa mutation, drug resistance factor (R-factor) plasmid, UV sensitivity, growth rate property, negative control value, positive control values, etc. The tester strains used in the study were verified to maintain specific characteristics.

Number of Plates
The number of plates used at each dose level was two in both the dose-finding test and the main test.

Test Procedures (Pre-incubation Method)
1) The test solution, vehicle or positive control article (0.1 mL of each) was placed into a sterilized small test tube, 0.5 mL of 0.1 mol/L phosphate buffer (pH 7.4) for the system without metabolic activation or 0.5 mL of S9 Mix for the system with metabolic activation was added, and then 0.1 mL, of bacterial solution was added to each tube.
2) Each mixture was pre-incubated while shaking (80 rpm) at 37OC for 20 minutes immediately after stirring, 2.0 mL of top agar kept at 4S°C was added to each tube, and this mixture was shaken and overlaid uniformly on the minimal glucose agar plate medium.
3) For a sterility test, 0.1 mL of the test solution at the highest dose or 0.5 mL, of S9 Mix was measured in a small test tube, and after 2.0 mL of top agar was added, it was overlaid uniformly on the minimal glucose agar plate medium. These processes, 1) to 3), were performed under fluorescent lamps with ultraviolet-absorbing films.
4) After verifying that the overlaid top agar was solidified, the minimal glucose agar plate medium was put upside down in an incubator and incubated at 37°C for 49 hours for the dose-finding test, for 48.5 hours for the main test.
5) After incubation, the culture was observed for the presence or absence of precipitation of the test article and coloration of the plate. Since neither precipitation nor coloration was observed at any dose levels with or without metabolic activation, the number of revertant colonies was counted with an automatic colony counter (Colony Analyzer CA-1 1D systems, System Science Co., Ltd.) (area correction, correction value: 1.21). The presence or absence of growth inhibition was observed using a stereoscopic microscope
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies to that of spontaneous revertant colonies (the negative control) and dose-response and reproducibility were noted, or even if no clear dose-response was observed but there was at least two-fold increase in comparison with the number of spontaneous revertant colonies and reproducibility was observed, the test article was judged to be positive.
Statistics:
Statistical method was not applied to the judgment

Results and discussion

Test results
Species / strain:
other: All tested strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
It was judged that this test was conducted appropriately since two-fold or more increase in the number of revertant colonies in comparison with the negative control was observed in the plate treated with the positive control article for each tester strain, the mean numbers of revertant colonies in the plates in the negative control group and positive control group were within the range of the control limit of background data, and no contaminants such as other bacteria were seen in this test system.

Observations of Dose-finding Test and Selection of Dose Levels for Main Test
To set the dose levels for the main test, the 50 mg/mL solution was diluted 6 times using a common ratio of 4 and a total of 7 dose levels (1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 ug/plate) were used in the dose-finding test.
In the dose-finding test, precipitation on the plate and coloration by the test article in the test systems were not observed at any dose levels with or without metabolic activation. In the observation of bacteria using a stereoscopic microscope, growth inhibition was observed at 313 ug/plate and above for S. typhimurium TA strains with or without metabolic activation and at 1250 ug/plate and above for E. coli WP2 uvrA with or without metabolic activation. There was neither increase more than two-fold in comparison with the number of spontaneous revertant colonies (the negative control value), nor dose-response in any test system with or without metabolic activation.
Therefore, using the minimum dose which showed growth inhibition as the maximum dose for the main test, the maximum dose level was set at 313 pg/plate for S. typhimurium TA strains with or without metabolic activation and at 1250 ug/plate for E. coli WP2 uvrA with or without
metabolic activation, and a total of 6 dose levels were selected by dilution 5 times at a common ratio of 2.

Observations of Main Test
Precipitation on the plate and coloration by the test article in the test systems were not observed at any dose levels with or without metabolic activation. In the observation of bacteria using a stereoscopic microscope, growth inhibition was observed at 156 ug/plate and above for S.
typhimurium TA98, TA100 and TA1537 with metabolic activation, at 313 ug/plate for S. typhirnurium TA1535 with metabolic activation and TA strains without metabolic activation and at 625 ug/plate and above for E. coli WP2 uvrA with or without metabolic activation.
There was neither increase more than two-fold in comparison with the number of spontaneous revertant colonies (the negative control value), nor dose-response in any test system with or without metabolic activation.

It was judged that this test was conducted appropriately since two-fold or more increase in the number of revertant colonies in comparison with the negative control was observed in the plate treated with the positive control article for each tester strain, the mean numbers of revertant colonies in the plates in the negative control group and positive control group were within the range of the control limit of background data (Attached Data), and no contaminants such as other bacteria were seen in this test system.

Discussion
In the dose-finding test and the main tests, there was neither increase more than two-fold in comparison with the spontaneous revertant colonies (the negative control value), nor dose response in any test system with or without metabolic activation.
Since more than two-fold increase in the number of revertant colonies in comparison with the negative control value was observed in the plate treated with the positive control article for each tester strain, it was judged that the reactions of the bacterial strains to the mutagenic agents were suitable and thus the study was conducted appropriately.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Based on the results of an AMES study conducted equivalent to OECD guideline 471 and GLP principes, 2-Hydroxy-benzoic acid 2-(1-methylethoxy) ethyl ester (Isopropoxyethyl salicylate) has no bacterial reverse mutagenic activity (without and with metabolic activation) under the conditions of this study.
Executive summary:

In order to examine the mutagenic potential of 2-Hydroxy-benzoic acid 2-(1-methylethoxy) ethyl ester(Isopropoxyethyl salicylate), a reverse mutation assay was conducted in Salmonella typhimurium TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2 uvrA with and without metabolic activation by the pre-incubation method. The study was performed equivalent to OECD guideline 471 and GLP principles. DMSO was used as the vehicle for the test article.

Precipitation on the plate and coloration by the test article in the test systems were not observed at any dose levels with or without metabolic activation. Testing was done up to and including cytotoxic concentratios. In the dose-finding test and main tests, there was neither more than two-fold increase in comparison with the number of spontaneous revertant colonies (the negative control value) nor dose-response in any test system with or without metabolic activation.

In conclusion, 2-Hydroxy-benzoic acid 2-(1-methy1ethoxy)ethyl ester (Isopropoxyethyl salicylate) was found to have no bacterial reverse mutagenic activity without and with metabolic activation under the conditions of this study.