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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

sodium 1,5-dioxo-1,5-bis({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy})-3-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)pentane-2-sulfonate

EC Number:

700-541-9

Cas Number:

1472634-24-4

Molecular formula:

C27H34F15NaO12S

IUPAC Name:

sodium 1,5-dioxo-1,5-bis({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy})-3-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)pentane-2-sulfonate

Test material form:

solid

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

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.