Sodium 6,14-diethyl-1,1,1,2,2,18,18,19,19,19-decafluoro-8,12-dioxo-10-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)-4,7,13,16-tetraoxanonadecane-9-sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, gene mutation in bacteria, OECD 471, GLP, Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA with and without S9: negative

Key, micronucleus in CHO, similar to OECD 487, non-GLP, with and without S9: negative

Key, chromosomal aberration, OECD 473, GLP, with and without S9: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 SEP 2015 - 26 APR 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: primary human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Primary human lymphocytes from blood from three healthy, non-smoking male volunteers from a panel of donors
- Suitability of cells: The use of human peripheral blood lymphocytes is recommended
- Normal cell cycle time (negative control): The measured cell cycle time of the donors used falls within the range 13 ± 2 hours.

For lymphocytes:
- Sex, age and number of blood donors: 3 male donors were used for the Range-Finder study (age: 29, 33, 30) and the chromosome aberration study (age: 27, 33, 29)
- Whether whole blood or separated lymphocytes were used: Whole blood cultures of pooled heparinised blood were established
- Whether blood from different donors were pooled or not: Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
- Mitogen used for lymphocytes: The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2 % of culture.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. Cultures were incubated at 37 ± 1°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9)
- source of S9 :
S9 was obtained from Molecular Toxicology Incorporated, USA
- method of preparation of S9 mix :
S9 is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolTox(TM) S-9 were stored frozen in aliquots at <-50°C prior to use. The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of the test article in a total of 10 mL.
- concentration or volume of S9 mix and S9 in the final culture medium:
The final concentration of liver homogenate in the test system was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Concentrations selected for the Chromosome Aberration Experiment were based on the results of this cytotoxicity Range-Finder Experiment. The concentrations in the Chromosome Aberration Experiment were as follows:
3h treatment + 17h recovery:
0, 10, 20, 40, 70, 100, 120, 140, 160, 180, 200, 225, 250 µg/mL (-S9)
0, 20, 40, 70, 100, 150, 200, 250, 300, 330, 360, 390, 420, 450, 500 µg/mL (+S9)
20h treatment + 0h recovery:
0, 5, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100 µg/mL (-S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is recommended as a well established organic solvent solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates for test article and positive controls, 4 replicates for vehicle control
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Blood cultures were incubated at 37 ± 1°C for approximately 48 hours and rocked continuously.
- Exposure duration/duration of treatment: either 3 h treatment in the absence and presence of metabolic activation or 20 h treatment in the absence of metabolic activation
- Harvest time after the end of treatment (sampling/recovery times): either 3 h treatment +17 h recovery or 20 h treatment +0 h recovery

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure:
Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 μg/mL to arrest dividing cells in metaphase.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
At the defined sampling time cultures were centrifuged at approximately 300 g for 10 minutes; the supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37 ± 1ºC for 15 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2 - 3 minutes) until the cell pellets were clean. Lymphocytes were kept in fixative at 2 - 8ºC prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, 2 - 3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading and several drops of suspension were transferred on to clean microscope slides labelled with the appropriate study details. Slides were flamed, as necessary, to improve quality. Slides were dried on a hot plate (set to approximately 80 - 100°C), then stained in filtered 4 % (v/v) Giemsa in pH 6.8 Gurr’s buffer for 5 minutes. The slides were rinsed, dried and mounted with coverslips using DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Three hundred metaphases per concentration (150 metaphases from each code) were analysed for chromosome aberrations. Where 15 cells with structural aberrations (excluding gaps) were noted on a slide, analysis may have been terminated.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The highest concentration selected for micronucleus analysis following all treatment conditions was one at which 50 - 60 % mitotic inhibition was achieved.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Structural aberrations were classified according to the ISCN scheme (ISCN, 1995). Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid with no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment.
- Determination of polyploidy/ endoreplication: Any cell with more than 48 chromosomes (that is, polyploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)

Rationale for test conditions:

Concentrations selected for the Chromosome Aberration Experiment were based on the results of this cytotoxicity Range-Finder Experiment. In the cytotoxicity Range-Finder Experiment concentrations up to 2000 µg/mL were applied.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
1. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) at one or more concentrations was observed (p≤0.05)
2. The incidence of cells with structural aberrations (excluding gaps) at such a concentration exceeded the normal range in both replicate cultures
3. There was a concentration-related increase in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were to be dealt with on a case-by-case basis.

Statistics:

The proportion of cells with structural chromosome aberrations excluding gaps was compared with the proportion in vehicle controls by using Fisher’s exact test. In addition, a Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical vehicle control range.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p≤0.05 were accepted as significant.

Species / strain:

primary culture, other: primary human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the limit of cytotoxicity (< 50 % in top dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH, osmolality: Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations. Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment: No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder Experiment (2000 μg/mL), compared to the concurrent vehicle controls (individual data not reported).
- Precipitation and time of the determination: Precipitation was observed in the Range-Finder experiment at the end of the treatment incubation period and at harvest. No precipitation was noted in the chromosome aberration experiment.
- Definition of acceptable cells for analysis: Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Any cell with more than 48 chromosomes (that is, polyploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately.

RANGE-FINDING/SCREENING STUDIES (if applicable): Concentrations selected for the Chromosome Aberration Experiment were based on the results of this cytotoxicity Range-Finder Experiment. In the range-finder, cultures were treated with vehicle control or the test article in a concentration range of 7.256 to 2000 µg/mL for a period of 3 hours (+ 17h recovery) with and without S-9 and 20 hours (+ 0h recovery).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Appropriate negative (vehicle) control cultures were included in the test system in the Chromosome Aberration Experiment under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within the 95th percentile of the current historical vehicle control (normal) ranges. Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. All acceptance criteria were considered met and the study was accepted as valid.

For all test methods and criteria for data analysis and interpretation:
please refer to the attached pdf

Chromosome aberration test (CA) in mammalian cells:
please refer to the attached pdf
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps: Structural aberrations were classified according to the ISCN scheme (ISCN, 1995) . Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid with no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : It may be noted that zero aberrations (with and/or without gaps) were commonly seen in vehicle and treated cultures during scoring of 150 metaphases per culture (and 300 per concentration) during the course of this study
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : A small increase in polyploid cells (considered not biologically relevant) was observed in one vehicle control culture for the 20+0 hour treatment in the absence of S-9, but no increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were observed in any cultures treated with the test item in the absence and presence of S-9 under any treatment condition.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
please refer to the attached pdf

Conclusions:

It is concluded that the test item did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9.

Executive summary:

Study Design

The test item was tested in an in vitro chromosome aberration assay according to OECD 473 in compliance with GLP using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Chromosome Aberration Experiment, limited by cytotoxicity, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of the test item on the mitotic index. Chromosome aberrations were analysed at three or four concentrations

Results

Appropriate negative (vehicle) control cultures were included in the test system in the Chromosome Aberration Experiment under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within the 95th percentile of the current historical vehicle control (normal) ranges. Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9, respectively. Cells receiving these were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with the test item for 3+17 hours in the absence and presence of S-9 and for 20+0 hours in the absence of S-9 resulted in frequencies of aberrant cells that were similar to the concurrent vehicle controls at all concentrations analysed. The aberration frequencies (excluding gaps) were within the normal ranges in all treated cultures analysed under the three treatment conditions. A small increase in polyploid cells (considered not biologically relevant) was observed in one vehicle control culture for the 20+0 hour treatment in the absence of S-9, but no increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were observed in any cultures treated with the test item in the absence and presence of S-9 under any treatment condition.

Conclusion

It is concluded that the test item did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 APR 2012 - 29 JUN 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS Operon (Salmonella typhimurium), TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Standard I Nutrient Broth (Merck KGaA, Darmstadt, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Standard I Nutrient Broth (Merck KGaA, Darmstadt, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix obtained from rats pre-treated with Aroclor 1254
Type and composition of metabolic activation system:
- source of S9 : S9 is routinely prepared in laboratory. Male Wistar, HSdCpb:Wu rats from Harlan Winkelmann, Borchen, Germany, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (Merck KGaA, Darmstadt, Germany). The animals received drinking water and a standard diet ad libitum. The body weight of the animals used was 183 ± 6.39 g. About 16 hours before sacrifice, the rats remained without food. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of 0.15 M KCI per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about + 4 °C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.
- method of preparation of S9 mix: The mixture of S9 plus the added cofactors is termed S9 mix.
Liver homogenate (S9): 0.10 mL (1st series), 0.30 mL (2nd series)
MgCl2/KCI aqueous solution (0.4 M/1.64 M): 0.02 mL (1st series), 0.02 mL (2nd series)
Glucose-6-phosphate, disodium salt: 5 µmol (1st series), 5 µmol (2nd series)
NADP, disodium salt: 4 µmol (1st series), 4 µmol (2nd series)
Sodium phosphate buffer (0.2 M, pH 7.4): 0.50 mL (1st series), 0.50 mL (2nd series)
Ultra pure water: 0.38 mL (1st series), 0.18 mL (2nd series)
Thus, 10 % and 30 % S9 in the S9 mix are used in the 1st and 2nd test series, respectively.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix is added to each plate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene are used as an acceptance criterion for each S9-batch.

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580, and 5000 µg/plate
2nd series: 50.0, 88.9, 158, 281, 500, and 1580 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason, only the respective solvent controls are used as the negative control in these studies.

- Justification for percentage of solvent in the final culture medium: 10 µL test material is applied. Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 μL per plate for water and 10 μL per plate for DMSO, ethanol, acetone or other organic solvents.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: Daunomycin, 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none
- Exposure duration/duration of treatment: The incubation of plates was performed at (37 +/- 1) °C for 2 to 3 days.

Evaluation criteria:

Definitions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies Maximal Mean Number of Colonies over the Actual Solvent Control
(Solvent Control) (Test Material)
≤ 10 ≤ 9 ≥ 30
≤ 30 ≤ 19 ≥ 40
≤ 80 ≤ 29 ≥ 80
≤ 200 ≤ 49 ≥ 120
≤ 500 ≤ 99 ≥ 200
Assessment: "No Increase" "Clear Increase"

All further results, ranging between "no" and "clear", are assessed as "weak increases".

Interpretations:
A test material is defined as non-mutagenic in this assay if
- "no" or "weak increases" occur in the test series performed. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed and the results of each series be discussed case by case.

Statistics:

not performed

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentration higher or equal to 500 µg/plate

RANGE-FINDING/SCREENING STUDIES: Based on the results of the 1st experimental series the concentrations for the 2nd series were selected. Because of the precipitation characteristics of the test material in the first series, high concentrations were omitted in the second experimental series.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The negative control mutant frequencies were all in the regular range. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, 9-aminoacridine, and cumene hydroperoxide in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning.

Ames test:
- Individual plate counts : please refer to attached pdf
- Mean number of revertant colonies per plate and standard deviation : please refer to attached pdf

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Executive summary:

Study Design

This GLP study was performed according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1^st and 30 % in the 2^nd series, respectively.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 1580 µg/plate. Toxicity to the bacteria was not observed. Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In the 1^st series of experiments, performed with and without the addition of rat liver S9 mix (10 %) as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. In the 2^nd series performed with 30 % S9 mix, a dose-related weak increase in the number of revertant colonies with external metabolizing system in tester strain TA 100 was evident.  The maximal effect observed was however certainly below the criteria for a “clear increase” and occurred at the limit of precipitation. Therefore, these fluctuations are considered as not biological relevant and thus the test material was not mutagenic in this assay.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 JUN 2012 - 06 JUL 2012 (Amendment No. 1 added on 14 May 2013)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Screening test, non-GLP, but GLP-like study with excellent documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

no short exposure (3-6 h) without S9

Principles of method if other than guideline:

Method complies with OECD Guideline 487 but with a reduced treatment regimen in the absence of metabolic activation (only treatment for 24 h)

GLP compliance:

no

Remarks:

study was not im compliance with GLP but with good documentation.

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster cells obtained from Dr. S. Albertini, Hoffmann-La Roche, Basel, Switzerland, on May 27, 1997. Cells were stored as frozen stocks in liquid nitrogen.

For cell lines:
- Absence of Mycoplasma contamination: Each batch of frozen cells was checked for spontaneous micronucleus frequency and for absence of mycoplasma.
- Methods for maintenance in cell culture: Cells were cultured in supplemented Dulbecco's Minimal Essential Medium (DMEM).

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: DMEM culture medium was supplemented with L-glutamin (4 mM), sodium bicarbonate (0.375 %), anitbiotics (penicilin/streptomycin) and 10 % fetal calf serum (FCS).
All incubations were performed at 37 °C in a 4 - 5 % carbon dioxide atmosphere (100 % humidity).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
S9 mix from livers of rats pretreated with Aroclor 1254
- source of S9 :
The S9 preparation used in this study was carried out at the Institute of Toxicology, Merck KGaA, Darmstadt, on March 22, 2011. S9 is routinely prepared by the laboratory following the proposal of Ames et al. (1975). Instead of potassium chloride solution, they used Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES pH 7.4.
- method of preparation of S9 mix:
Male Wistar, HSdCpb:Wu rats from Harlan (Netherlands) aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (Merck, Darmstadt, Germany). The animals received drinking water and a standard diet ad libitum.
About 16 hours before sacrifice, the rats remained without food. On day 5 to 7, they were sacrificed, the livers removed and collected in ice-cooled sterilized beakers containing PBS-HEPES. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of PBS-HEPES per gram of liver wet-weight. After homogenization, the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about + 4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 fraction was then frozen and stored in liquid nitrogen at -196°C.
- concentration or volume of S9 mix and S9 in the final culture medium:
The final S9 concentration was 1%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The bacterial mutagenicity (according to Ames et al., 1975) of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is determined once for each S9 batch. Clear increases in the number of revertants for various bacterial strains with all positive controls are used as an acceptance criterion for each S9 batch. The acceptance criterion mentioned was perfectly met.

Test concentrations with justification for top dose:

15.8, 28.1, 50.0, 88.9, 158, 281, 500 and 1580 µg/mL
The test item did not precipitate in the culture medium up to the highest dose applied (1580 µg/mL). It was cytotoxic to the V79 cells at concentrations ≥ 281 µg/mL (without S9 mix) and ≥ 500 µg/mL (with S9 mix). Concentrations ranging from 28.1 µg/mL up to 281 µg/mL were therefore evaluated for the presence of micronuclei.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle/ Justification for percentage of solvent in the final culture medium: Acetone (0.1 % final concentration) was used as solvent for the test material. Analysis of the historical data of our laboratory and experience of other research groups showed that such amounts of the selected solvents have no influence on the micronucleus frequency in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: Griseofulvin

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10E4 cells were seeded in 5 mL culture medium per slide in Quadriperm® culture dishes and cultured for 6 hours.
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Cells were seeded and cultured for 6 hours prior to treatment.
- Exposure duration/duration of treatment: The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix.
- Harvest time after the end of treatment (sampling/recovery times): Preparation of cells was performed 24 hours after start of treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Preparation of cells was performed by use of a hypotonic 2.25% sodium citrate solution and a fixative consisting of a mixture of 100 mL concentrated acetic acid + 300 mL ethanol + 5 mL 37.5% formaldehyde. After fixation, slides were dried for at least 72 hours and then stained with May-Grünwald and Giemsa solutions. Stained slides were dried overnight and then made permanent with Entellan®.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 1000 cells per slide were scored
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): A total of 1000 cells per slide were scored for micronuclei using Zeiss light microscopes with plane optics. Round particles with less than the 3rd of the diameter of the main nucleus, were scored as micronuclei where main and micronuclei must be clearly separated from each other. Cells with fragmented nuclei were not counted.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: A total of 1000 cells per slide were scored for the occurrence of mitotic figures. The percentage of cells with normal cell nucleus and cells in mitosis was determined.

Rationale for test conditions:

At least three concentrations over an adequate concentration range should be employed. The highest concentration should precipitate in the culture medium or exhibit cytotoxicity. The cytotoxicity of the lowest concentration should usually correspond to that of the negative controls. Soluble chemicals, if not toxic, are tested up to a maximum concentration of 1580 µg/mL in our screening version of the micronucleus test. According to these criteria the concentrations were selected for the main study in the presence and absence of S9 mix.
In this study the test item did not precipitate in the culture medium up to the highest dose applied (1580 µg/mL). It was cytotoxic to the V79 cells at concentrations ≥ 281 µg/mL (without S9 mix) and ≥ 500 µg/mL (with S9 mix). Concentrations ranging from 28.1 µg/mL up to 281 µg/mL were therefore evaluated for the presence of micronuclei.

Evaluation criteria:

The assay is considered valid if:
- the mean micronucleus frequencies in the negative (solvent) control cultures fell within the normal range for this test system and
- at least one concentration of each of the positive control chemicals relevantly increased the mutation frequency as compared to the actual negative control (at least a 2-fold increase).

Test materials are assessed as negative or non-mutagenic in this test system if:
- the assay is considered valid and
- no relevant increase in the mutation frequency (at least a 2-fold) occurs.
Test materials are assessed as positive or mutagenic in this test system if:
- the assay is considered valid and
- a clear increase in the micronucleus frequency (at least a 3-fold) occurs dose-dependently (over at least two test material concentrations) or reproducibly at identical concentrations in two independent experimental series performed and
- the maximal test material-induced value is above the highest value of the historical negative control value

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

281 µg/mL without S9, 500 µg/mL with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
A Screening test (Study no. T18361) was performed with and without metabolic activation. The test material dissolved in acetone was tested at 28.1, 50.0, 88.9, 158 and 281 µg/mL without S9 mix and at 88.9, 158, 281 and 500 µg/mL with S9 mix. Griseofulvin (GRIS) was tested as positive control without metabolic activation, 7,12-Dimethylbenz(a)anthracene (DMBA) as positive control with metabolic activation. No mutagenic potential was concluded from the screening test system.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The level of micronucleus frequencies of the negative and positive controls observed in the current experiment matched those determined as historical controls of the performing laboratory. The study was therefore accepted as valid.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
please refer to attached pdf

- Genotoxicity results
please refer to attached pdf

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: please refer to attached pdf

Conclusions:

The test item was not mutagenic in this screening test system with and without metabolic activation (S9 mix).

Executive summary:

The registered substance was tested in a non-GLP screening assay similar to OECD Guideline 487 but with reduced treatment regimen.

The test material was dissolved in acetone and screened for its ability to induce micronuclei in V79 Chinese hamster cells. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The concentrations tested were selected on the basis of the cytotoxicity characteristics of the test material. The test material did not precipitate up to the highest concentration applied (1580 µg/mL). It was cytotoxic to the V79 cells at concentrations of ≥ 281 µg/mL (without S9 mix) and ≥ 500 µg/mL (with S9 mix). Concentrations ranging from 28.1 µg/mL up to 281 µg/mL were therefore evaluated for the presence of micronuclei. Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S9 mix.

Micronucleus frequencies in negative control cultures fell within normal ranges, and clear increases were induced by the positive control chemicals. Therefore, the study was accepted as valid.

No relevant increases in micronucleus frequency were observed following treatment of the cells with the test material neither in absence nor in presence of S9 mix up to the limit of cytotoxicity. It is therefore concluded that the test material is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria, OECD 471

Study Design

This GLP study was performed according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1^st and 30 % in the 2^nd series, respectively.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 1580 µg/plate. Toxicity to the bacteria was not observed. Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene was used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In the 1^st series of experiments, performed with and without the addition of rat liver S9 mix (10 %) as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. In the 2^nd series performed with 30 % S9 mix, a dose-related weak increase in the number of revertant colonies with external metabolizing system in tester strain TA 100 was evident.  The maximal effect observed was however certainly below the criteria for a “clear increase” and occurred at the limit of precipitation. Therefore, these fluctuations are considered as not biological relevant and thus the test material was not mutagenic in this assay.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

In a supporting study, the test item was also not mutagenic in an Ames screening test in Salmonella typhimurium strain TA 102.

Micronucleus in CHO, similar to OECD 487

Study Design

The registered substance was tested in a non-GLP screening assay similar to OECD Guideline 487 but with reduced treatment regimen.

The test material was dissolved in acetone and screened for its ability to induce micronuclei in V79 Chinese hamster cells. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix. The concentrations tested were selected on the basis of the cytotoxicity characteristics of the test material. The test material did not precipitate up to the highest concentration applied (1580 µg/mL). It was cytotoxic to the V79 cells at concentrations of ≥ 281 µg/mL (without S9 mix) and ≥ 500 µg/mL (with S9 mix). Concentrations ranging from 28.1 µg/mL up to 281 µg/mL were therefore evaluated for the presence of micronuclei. Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S9 mix.

Results

Micronucleus frequencies in negative control cultures fell within normal ranges, and clear increases were induced by the positive control chemicals. Therefore, the study was accepted as valid.

No relevant increases in micronucleus frequency were observed following treatment of the cells with the test material neither in absence nor in presence of S9 mix up to the limit of cytotoxicity.

Conclusion

It is therefore concluded that the test material is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Chromosomal aberration, OECD 473

Study Design

The test item was tested in an in vitro chromosome aberration assay according to OECD 473 in compliance with GLP using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Chromosome Aberration Experiment, limited by cytotoxicity, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of the test item on the mitotic index. Chromosome aberrations were analysed at three or four concentrations

Results

Appropriate negative (vehicle) control cultures were included in the test system in the Chromosome Aberration Experiment under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within the 95th percentile of the current historical vehicle control (normal) ranges. Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9, respectively. Cells receiving these were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with the test item for 3+17 hours in the absence and presence of S-9 and for 20+0 hours in the absence of S-9 resulted in frequencies of aberrant cells that were similar to the concurrent vehicle controls at all concentrations analysed. The aberration frequencies (excluding gaps) were within the normal ranges in all treated cultures analysed under the three treatment conditions. A small increase in polyploid cells (considered not biologically relevant) was observed in one vehicle control culture for the 20+0 hour treatment in the absence of S-9, but no increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were observed in any cultures treated with the test item in the absence and presence of S-9 under any treatment condition.

Conclusion

It is concluded that the test item did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to the limit of cytotoxicity for 3+17 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 20+0 hours in the absence of S-9.

Overall conclusion

The test material was not mutagenic in assays in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA in the presence or absence of exogenous metabolic activation system.

The test material was not mutagenic in the micronucleus test in Chinese hamster ovary cells with and without metabolic activation.

Furthermore, the chromosome aberration test in human peripheral lymphocytes showed no increase in structural chromosomal aberrations in the presence or absence of metabolic activation.

Based on these negative in vitro findings no further in vivo investigations are required.

Justification for classification or non-classification

The key studies indicate no genotoxic potential present in vitro, thus no classification for mutagenicity is triggered in accordance with Regulation (EC) No 1272/2008.