2-isopropoxyethyl salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The conclusion on genetoxic properties of Sakura Salicylate is based on the results of three studies, which together cover all potential mode-of-actions. All three studies were performed according to OECD guidelines and GLP principles (Klimisch 1).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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

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

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

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

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'.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2015 - 31 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro chromosome aberration assay

Target gene:

The test item SAKURA SALICYLATE was assayed for the ability to induce chromosomal aberrations in Chinese hamster ovary cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates from rats pre-treated with Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

Main assay 1 -S9 12.9, 6.46 and 3.23 ug/mL
Main assay 1 +S9 188, 85.5 and 38.8 ug/mL
Main assay 2 - S9 8.49, 14.9 and 26.0 ug/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent:This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Statistically significant increases in the number of aberrant cells were observed following treatments with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system.

Details on test system and experimental conditions:

Two main experiments were performed. In the first main experiment, the cells were treated for 3 hours both in the presence and absence of S9 metabolism.
The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used.
As negative results were obtained in the first main experiment, a second main experiment was performed in the absence of S9 metabolism using
a continuous treatment until harvest at 20 hours.
Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.
Following treatment, the pH and osmolality of the treatment media at higher dose levels were determined for all treatment series.

Colcemid was added (0.2 µg/mL final concentration) for the last three hours of the treatment period, leading up to harvesting. Cells were harvested at
20 hours after beginning of treatment.

At harvesting the medium was removed from the flasks and the cells were brought into suspension by trypsinization. The cell suspension was centrifuged and the cell pellet was resuspended in hypotonic solution.
The cells were then fixed in freshly prepared methanol:acetic acid fixative and washed with fixative. A few drops of the cell suspension obtained in this waywere dropped onto slides to produce metaphase chromosome spreads. For each culture three slides were prepared. They were stained in 3% Giemsa
and then made permanent with Eukitt.

Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of population doubling.

For the three selected doses, for the solvent and for the positive controls, 150 metaphase spreads per cell culture were scored to assess the frequency of aberrant cells. Polyploid and endoreplicated cells were also recorded.

Evaluation criteria:

In this assay, the test item is considered as clearly positive if the following criteria are met:

– Any dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps).
– The incidence of cells bearing aberrations is outside the normal distribution of historical control values.
– The increase of cells bearing aberration is dose-related when evaluated with an appropriate trend test.

The test item is considered clearly negative in this assay if none of the above criteria is met.

Statistics:

Fisher’s Exact Test was used to compare the number of cells bearing aberrations in control and treated cultures.
Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps.
Cochran-Armitage trend test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

Following treatment with the test item, no precipitation or opacity was observed at the beginning or at the end of treatment, in the absence or presence of S9 metabolism in any experiment. No remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism.
Following treatment with the test item, no increase in the incidence of cells bearing structural aberrations, over the concurrent control value was observed in the presence or absence of S9 metabolism, at any concentration in any experiment. Increases in the incidence of endoreduplicated cells over the control value, more marked at the intermediate dose level, were observed in the presence of S9 metabolism. For this test point the increase was not reproducible in replicate cultures, however the increase reached statistical significance (p<0.05) for one culture and for both cultures combined, indicating that the test item might have the potential to inhibit cell cycle progression.
The incidence of endoreduplicated cells in the negative controls was slightly higher than the maximum acceptable limit based on historical control values.
Marked increases in the incidence of cells bearing aberrations were observed following treatments with the positive controls Cyclophosphamide andMitomycin-C, indicating the correct functioning of the test system.

Conclusions:

Based on the results of a chromosome aberration test, performed according to OECD guideline and GLP principles, it is concluded that Sakura Salicylate does not induce structural aberrations in mammalian cells cultured in vitro.

Executive summary:

A chromosome aberration test was performed with Sakura Salicylate according to OECD guideline and GLP principles. The proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data. Adequate number of cells (at least 300 at each test point) and test item concentrations were analysable. The positive control items, Mitomycin-C and Cyclophosphamide, induced statistically significant increases in the incidence of cells with structural aberrations compared with the concurrent negative control and the responses were compatible with the historical control range. The study was thus accepted as valid.

No statistically significant increase in the incidence of cells bearing aberrations (including or excluding gaps) was observed at any dose level and treatment series and no statistically significant dose effect relationship was observed in any treatment series. The incidences were inside the normal distribution of historical control values. Increases in the incidence of endoreduplicated cells over the control value, more marked at the intermediate dose level, were observed in the presence of S9 metabolism. For this test point the increase was not reproducible in replicate cultures, however the increase reached statistical significance (p<0.05) for one culture and for both cultures combined, indicating that the test item might have the potential to inhibit cell cycle progression.

Overall it is concluded that there are no indications that Sakura Salicylate induces structural aberrations in mammalian cells cultured in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue
trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid
synthesis resulting in the death of TK-competent cells.
TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its
presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK
locus (TK+/-) may undergo a single step forward mutation to the TK+/- genotype in which little or no TK activity remains.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated
that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect
not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 10% Foetal Calf Serum (RPMI Complete)
- Properly maintained: yes; permanente stocks are stored in liquid idrogen and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
-The generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Species Rat Strain Sprague Dawley Tissue Liver Inducing Agents Phenobarbital – 5,6-Benzoflavone Producer MOLTOX, Molecular Toxicology, Inc. Batch Number 3417

Test concentrations with justification for top dose:

A preliminary solubility trial indicated that the test item was soluble at 224 mg/mL in DMSO. This concentration was equivalent to 2240 μg/mL in the
final treatment medium, corresponding to the highest concentration to be tested, 0.01M.
On the basis of this result, a concentration of 2240 μg/mL was selected as the top dose level to be used in the cytotoxicity test. Both in the absence and
presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2240 μg/mL and at a wide range of lower dose levels: 1120, 560,
280, 140, 70.0, 35.0, 17.5 and 8.75 μg/mL.
Two independent assays for mutation at the TK locus were performed using the dose levels described below:
Main Assay I (-S9, treatment time 3 hours): 280, 200, 140, 70.0, 35.0 and 17.5 μg/mL
Main Assay I (+S9, treatment time 3 hours): 510, 378, 280, 140, 70.0 and 35.0 μg/mL
Main Assay II (-S9, treatment time 24 hours): 200, 167, 139, 69.4, 34.7 and 17.4 μg/mL
Main Assay II (+S9, treatment time 3 hours): 370, 336, 306, 278, 139 and 69.4 μg/mL

Vehicle / solvent:

Test item solutions were prepared using DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activatio. A single culture was used at each test point.
The mutation assays were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and incubated to allow expression of the mutant phenotype. At the end of the expression period cells were plated for the evaluation of 5-trifluorothymidine resistance and for viability.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126x10-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria will be dealt with on a
case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological
significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Survival after treatment: In the first experiment, in the absence of S9 metabolic activation, no cells survived to treatment at the highest dose level tested (280 μg/mL). At the two next lower dose levels (200 and 140 μg/mL), dose-related toxicity was observed, reducing relative total growth (RTG) to 21 and 63% of the concurrent negative control, respectively. No toxicity was observed over the remaining dose levels tested. In the presence of S9 metabolic activation, no cells survived to treatment at the highest dose level tested (510 μg/mL).
Severe toxicity, reducing RTG to 12% of the concurrent negative control value, was observed at 378 μg/mL. No toxicity was observed over the remaining dose levels tested.
In the second experiment, in the absence of S9 metabolic activation using a long treatment time, dose-related toxicity was observed at the three highest dose levels reducing RTG to 32, 64 and 76% of the concurrent negative control value. No toxicity was observed at the remaining concentrations. In the presence of S9 metabolism, severe toxicity was noted at the highest dose level (370 μg/mL) reducing RTG to 2%. At the two next lower dose levels (336 and 306 μg/mL), dose related toxicity was observed, reducing relative total growth (RTG) to 43 and 72% of the cuncurrent negative control, respectively. No toxicity was observed over the remaining dose levels tested.
At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, we have excluded from the statistical analyses, mutation data obtained in the presence of S9 metabolism at 370 μg/mL in the second experiment.
Mutation results: No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level.
For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.
The pH values and osmolality of the post-treatment media were determined. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Conclusions:

Based on the results of an MLA study performed according to OECD guidelines and GLP principles, it is concluded that under the reported experimental conditions Sakura Salicylate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.

Executive summary:

Sakura Salicylate was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the test item was soluble at 224 mg/mL in DMSO. This concentration was equivalent to 2240 μg/mL in the final treatment medium, corresponding to the highest concentration to be tested, 0.01M. Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed. Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that Sakura Salicylate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

A chromosome aberration test was performed with Sakura Salicylate according to OECD guideline and GLP principles. The proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data. Adequate number of cells (at least 300 at each test point) and test item concentrations were analysable. The positive control items, Mitomycin-C and Cyclophosphamide, induced statistically significant increases in the incidence of cells with structural aberrations compared with the concurrent negative control and the responses were compatible with the historical control range. The study was thus accepted as valid. No statistically significant increase in the incidence of cells bearing aberrations (including or excluding gaps) was observed at any dose level and treatment series and no statistically significant dose effect relationship was observed in any treatment series. The incidences were inside the normal distribution of historical control values. Increases in the incidence of endoreduplicated cells over the control value, more marked at the intermediate dose level, were observed in the presence of S9 metabolism. For this test point the increase was not reproducible in replicate cultures, however the increase reached statistical significance (p<0.05) for one culture and for both cultures combined, indicating that the test item might have the potential to inhibit cell cycle progression. Overall it is concluded that there are no indications that Sakura Salicylate induces structural aberrations in mammalian cells cultured in vitro.

Sakura Salicylatewas examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the test item was soluble at 224 mg/mL in DMSO. This concentration was equivalent to 2240 μg/mL in the final treatment medium, corresponding to the highest concentration to be tested, 0.01M. Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed. Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded thatSakura Salicylate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

Based on the available data, it is concluded that there are no indications for genotoxic properties of Sakura Salicylate. Therefore the substance is not classified for genotoxic properties according to Regulation (EC) 1272/2008.