Strontium 3-hydroxy-4-[(1-sulfonato-2-naphthyl)azo]-2- naphthoate 5/2 hydrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was considered that R507-2 did not induce the chromosomal aberration under the present test conditions.

It is concluded that R507-2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 6, 2007 - January 24, 2008

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

625, 1250, 2500 and 5000 µg/mL in short-term treatments without and with S9 mix.
78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL in 24 hours continuous treatment.

In the cell growth inhibition test, for the short-term treatments without and with S9 mix, the highest dose of the test substance was selected at 5000 µg/mL and the following 3 doses were set based on a geometric progression of 2, because a cytotoxicity such that the cell growth rate was less than 50% was not obtained. For 24 hours continuous treatment, the highest dose of the test substance was selected at dose of over IC50, because a cytotoxicity that the cell growth rate was less than 50% was obtained. Though IC50 was calculated at 1300 µg/mL, the cell growth rate was 32.2% at 5000 µg/mL. Therefore, the highest dose was selected at 5000 µg/mL and the following 6 doses were set based on a geometric progression of 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle:In the result of solubility test of the test substance, the test substance was not dissolved at 50.0 mg/mL in distilled water and at 500 mg/mL in DMSO or acetone. The results showed that the test substance suspension at 50.0 mg/mL in 0.5 w/v % CMC solution was homogeneous state. This suspension was not indicated any change in color nor exothermic within 2 hours after preparation. Therefore, 0.5 w/v % CMC solution was selected in a solvent.

Positive controls:

yes

Positive control substance:

other: mitomycin c (MMC), cyclophosphamide monohydrate (CPA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);
Chromosomal aberration test was carried out using the same procedure as that of the cell growth inhibition test, with the following positive controls. In the preparation of specimens, the cells were completely fixed by 2 to 5 changes with 3 mL of fixative solution. Two specimens per dish (four specimens per dose) were prepared.
In the cell growth inhibition test for 24 hours continuous treatment, it was difficult
to analyze the chromosomes at the doses up 313 µg/mL by precipitation of the test substance, therefore, the medium was exchanged using the same procedure as that of the short-term treatment before the demecolcine solution was added at 2 hours before the end of the culture
In the positive control, after a same volume of distilled water as that of the test substance formulation was added in the treatment medium, each dish was added with 30 µL of a 0.01 mg/mL MMC solution and 18 µL of a 1 mg/mL CPA solution for the short-term treatments without and with S9 mix, respectively, and 25 µL of a 0.01 mg/mL MMC solution for the continuous treatment.

Evaluation criteria:

The findings were judged to be positive when the frequencies of cells with structural aberrations showed 10 % or more with a dose-related increase or the frequencies of cells with structural aberrations showed 5 % or more both in the chromosomal aberration test and the confirmation test. The other cases were judged to be negative. No statistical analyses were used. The frequency of numerical aberration cells was judged according to the same criteria as that of the structural aberration.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Short-term Treatment
1) Without S9 mix
Cell growth rate and IC50
The cell growth rates at 625, 1250, 2500 and 5000 µg/mL of the test substance were 90.1, 91.9, 85.6 and 75.7 %, respectively. The IC50 was calculated more than 5000 µg/mL.
(2) Precipitation of the test substance, color change of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end
of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
(3) Frequency of cells with structural aberrations
The frequencies of cells with structural aberrations were 3.0 % in the negative control and 63.0 % in the positive control. The frequencies of cells with structural aberrations at 1250, 2500 and 5000 µg/mL were and 0.5, 0.5 and 0.0 %, respectively, therefore, the results were judged to be negative.
(4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were Jess than 5 % at all doses including the negative and positive controls, therefore, the results were judged to be negative.

2) With S9 mix
(1) Cell growth rate and IC50
The cell growth rates at 625, 1250, 2500 and 5000 µg/mL of the test substance were 84.0, 83.7, 75.5 and 73.1 %, respectively. The IC50 was calculated more than 5000 µg/mL.
(2) Precipitation of the test substance, color change of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
(3) Frequency of cells with structural aberrations
The frequencies of cells with structural aberrations were 2.5 % in the negative control and 33.0 % in the positive control. The frequencies of cells with structural aberrations at 1250, 2500 and 5000 µg/mL were 2.5, 1.5 and 3.0 %, respectively, therefore, the results were judged to be negative.
(4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were Jess than 5% at all doses including the negative and positive controls, therefore, the results were judged to be negative.
Twenty Four Hours Continuous Treatment
1) Cell growth rate and IC50
The cell growth rates at 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL of the test
substance were 104.7, 103.6, 89.9, 77.7, 42.6, 24.9 and 18.0 %, respectively. The IC50 was calculated at 1100 µg/mL.
2) Precipitation of the test substance, color of medium and corrosion of culture dish
Precipitation of the test substance was observed at all doses at the start and the end of the treatment and at the end of the culture. The color change of the medium and the corrosion of the culture dish were not observed at all doses.
3) Frequency of cells with structural aberrations
The frequencies were 1.5 % in the negative control and 62.5 % in the positive control. The frequencies of cells with structural aberrations at 313, 625 and 1250 µg/mL were 1.0, 1.5 and 2.0 %, respectively, therefore, the results were judged to be negative.
4) Frequency of numerical aberration cells
The frequencies of numerical aberration cells were less than 5.0 % at all doses including the negative and positive controls, therefore, the results were judged to be negative.

Conclusions:

In each treatment method in the chromosomal aberration test, the frequencies of cells with aberrations were less than 5 % in the negative controls, and the frequencies of cells with structural aberrations excluding gaps were more than 20 % in the positive controls, indicating that the present study was appropriately performed.
As a result of chromosomal aberration test, the frequencies of cells with structural aberration and numerical aberration cells showed less than 5 % at all doses of the test substance in the short-term treatments without and with S9 mix and 24 hours continuous treatment, therefore both of structural aberration and numerical aberration were judged to be negative.
Based on the above results, it was considered that R507-2 did not induce the chromosomal aberration under the present test conditions.

Executive summary:

The ability of R507-2 to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cells).

Based on the result of cell growth inhibition test, the doses in the chromosomal aberration test were set at 625, 1250, 2500 and 5000 µg/mL in short-term treatments without and with S9 mix and at 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL in 24 hours continuous treatment.

In the short-term treatments without and with S9 mix, the observation doses of specimens were selected at 1250, 2500 and 5000 µg/mL as the higher 3 setting doses, because a cytotoxicity such that the cell growth rate was less than 50 % was not obtained. In 24 hours continuous treatment, the observation doses of specimens were selected at 313, 625 and 1250 µg/mL, because cell growth rate of 1250 µg/mL was less than 50 %.Inthe observation, the frequencies of cells with structural aberrations and numerical aberration cells were scored.

As a result of observation of specimens, the frequencies of cells with structural aberration and numerical aberration cells showed less than 5 % at all observation doses of the test substance in all treatment methods, therefore both of structural aberration and numerical aberration were judged to be negative.

On the other hand, the frequencies of cells with structural aberrations or numerical aberration cells in the negative control treated with 0.5 w/v % carboxymethyl cellulose sodium salt solution showed less than 5 %, and the frequencies of cells with structural aberrations in the positive controls treated with mitomycin C or cyclophosphamide monohydrate, showed more than 20 %, indicating the proper performance of the present study.

It was concluded that R507-2 did not induce the chromosomal aberration under the present test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2007 - 29 November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the direct plate assay. The highest concentration of R507-2 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 10, 33, 100, 333 and 1000 µg/plate.

Vehicle / solvent:

The test substance was suspended in dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany). Preparation of test solutions started with solutions of 50 mg/mL for the dose range finding and of 10 mg/mL for the mutation experiments. The stock solutions were treated with ultrasonic waves to obtain a homogeneous suspension. The lower test concentrations were prepared by subsequent dilutions in DMSO. Test substance concentrations were used within 1.5 to 4 hours after preparation.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Source: Recommended test system in international guidelines (e.g. OECD, EEC).

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (1.0E9 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi). The agar plates for the test with the Salmonella typhimurium strains also contained
12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) Sodium Chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in the dark at 35.7 - 38.9°C (protocolled range 37.0 ± 1.0°C). Temporary deviations of maximally 1 hour (in the range of 34.0 - 38.5°C) occurred due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EEC).

Evaluation criteria:

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2u vrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

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:

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:

E. coli WP2 uvr A

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

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that R507-2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of R507-2 in the Salmonella typhimurium reverse mutation

assay and the Escherichia colir everse mutation assay (with independent repeat).

 

R507-2 was tested in the Salmonella typhimurium reverse mutation assay with four histidine­ requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed with rat liver S9-mix (rat liver S9-mix induced by a combination of phenobarbital and P-naphthoflavone) and secondly a preincubation assay was performed with hamster liver S9-mix(uninducedmaleGoldenSyrianHamsterliverS9-mix).

 

The study procedures described in this report were based on the most recent OECD and EEC guidelines.

 

Batch 070711 of R507-2 was a red powder with a purity of 96.4%.The test substance was suspended in dimethyl sulfoxide.

 

In the direct plate assay, R507-2 was initially tested in a dose range finding study up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA. R507-2 precipitated on the plates at dose levels of 1OOOµg/plate and above. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Secondly, R507-2 was tested up to concentrations of 1000 µg/plate in the strains TA1535, TA1537 and TA98. R507-2 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the preincubation assay, R507-2 was tested up to concentrations of 1OOOµg/plate in the testerstrainsTA1535,TA1537, TA98,TA100andWP2uvrA.R507-2 precipitated on the plates at dose levels of 333 and 1OOOµg/plate in the absence of S9 -mix and at 1OOOµg/plate in the presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

R507-2 did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9- metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that R507-2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

genetic toxicity in vitro, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2017 - 14 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows: 0.03, 0.06, 0.13, 0.25, 0.5 ,1 (μg/mL)

Vehicle / solvent:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. Acetone is toxic to L5178Y cells at dose volumes greater than 0.5 % of the total culture volume. The molecular weight of the test item was 553.1. Therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL initially, the maximum recommended dose level, and a correction for the purity of the test item was applied. The test item formed a suspension unsuitable for dosing at 200 mg/mL. Therefore, the test item was formulated at 100 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 500 μg/mL in the subsequent preliminary toxicity test. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at eight dose levels of the test item (0.03 to 4 μg/mL for all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency:
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data, a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Concentration of R507-2 (μg/mL) plated for viability and mutant frequency: 0.03, 0.06, 0.13, 0.25, 0.5 ,1

Results

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

All three in-vitro studies were concluded as non-mutagenic therefore we can confirm that R507-2 does not cause genetic toxicity. Therefore according to the CLP regulation the test substance is classified as non-hazardous for genetic toxicity.