2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

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:

17 August 2018 to 19 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

EC Number:

260-828-5

EC Name:

2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Cas Number:

57583-34-3

Molecular formula:

C31H60O6S3Sn

IUPAC Name:

2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate

Test material form:

liquid

Details on test material:

- Physical appearance: Yellow liquid
- Storage conditions: Ambient (21 to 29 °C)

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: Human peripheral lymphocytes from the blood of healthy, young, non-smoking donors, 24 (Male), 31 (Male) and 25 (Male) years of age with no known recent exposure to genotoxic chemicals or radiation were used. Blood from each individual was collected in a sodium heparin vacutainer and analysed using Advia 2120.
Blood samples were collected using sterile syringe, transferred to sodium heparin and stored at 2 to 8º C till usage.
- Suitability of cells: As all the parameters were in acceptable range, blood was used from a single donor for initial cytotoxicity and chromosomal aberration test. A consent form was received from the volunteer for the donation of the blood. The blood sample obtained was subjected for evaluation in a haematology analyser. The sample was accepted as the results were within the in-house acceptable range.
Quantity of 50 µL of each test material dilutions of concentrations 3.125, 6.25, 12.5, 25, 50, 100 and 200 µL/mL was mixed with media and made upto 5 mL to get concentrations of 0.0312, 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL along with vehicle control. The contents were incubated at 37 ± 1 ºC with 5 ± 1 % CO2. Results were recorded after 24 hours of incubation for change in pH and signs of precipitation.
- Modal number of chromosomes: The chromosome number of human lymphocytes is 2n=46. Since, fixation procedures often result in the breakage of a proportion of metaphase cells with loss of chromosomes, the cells with 46 ± 2 number of chromosomes were considered for analysis.

MEDIA USED
- Type and composition of media: Culture Media: RPMI Media supplemented with 10 % FBS and antibiotics (1 % Penicillin-Streptomycin) was used. The pH of the culture medium used was 7.32 to 7.40. The media was stored at 2 to 8 ºC till use and was thawed to room temperature before use.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar Rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice.
- Method of preparation of S9 mix: The S9 homogenate was prepared and stored in the test facility at -80 ± 10 ºC until use. 1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.29 for initial cytotoxicity test, 7.29 for follow up cytotoxicity and 7.32 for chromosomal aberration test.
- Quality controls of S9: The batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolise the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.

Test concentrations with justification for top dose:

The following set of criteria was followed for the selection of concentrations for chromosomal aberration test:
- Three analysable concentrations were used for chromosomal aberration test.
- Based on the results of follow-up cytotoxicity test, the concentrations selected for the chromosomal aberration test were 0.0312, 0.0625, 0.125 µL /mL as low, mid and high concentrations respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was found miscible in DMSO at 200 µL/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Methods of slide preparation: Pellets were mixed with 3 mL of freshly prepared warm 0.56 % Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre-labelled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5 % Giemsa stain for 15 minutes.
All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
Gaps were recorded separately and reported but not included in the total aberration frequency.
Coding of slides was not carried out for initial cytotoxicity test. For each replicate a minimum of 500 cells were scored.

Percent mitotic index (MI %) was determined by the following formula:

Mitotic Index MI% = (Number of Mitotic cells / Total number of cells scored) × 100

Percent reduction in mitotic index was obtained by using the formula:

= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100

VC: Vehicle Control
MI: Mitotic Index.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Initial Cytotoxicity Test for Selection of Test Concentrations: Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test. The concentrations selected for initial cytotoxicity test were 0.25, 0.5, 1 and 2 µL/mL.
- Initial and Follow up Cytotoxicity Test Procedure: Whole blood of volume 0.5 mL was added to each tube containing culture media of volume 4.5 mL supplemented with 2 % phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37 ± 1 ºC and 5 ± 1 % CO2.
Post 44 to 48 hours of incubation with PHA, cells from tubes were centrifuged at 1 500 rpm for 10 minutes, supernatant was discarded.
Cell pellet was resuspended with 2 to 3 mL fresh media.
For tubes with metabolic activation (+S9) - set 1, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle, 0.5 mL of S9 mix and volume was made up to 5 mL with culture media.
For tubes without metabolic activation (-S9) - set 2 and 3, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle and the volume was made up to 5 mL with culture media.
All the test material concentrations and controls were maintained in duplicate.
Cells were incubated both with metabolic activation (+S9) - set 1 for 3 to 6 hours, without metabolic activation (-S9) - set 2 for 3 to 6 hours and without metabolic activation (-S9) for 20 to 24 hours Set 3 at 37 ± 1 ºC and 5 ± 1 % CO2.
The treatment for set 1 and 2 tubes were terminated post 3 to 6 hours of incubation, by centrifugation at 1 500 rpm for 10 minutes.
Supernatant was discarded and cell pellet was mixed with 5 mL of culture medium and incubated further to complete 20 to 24 hours starting from the start of treatment.
2 hours (For Intial cytotoxicity test, follow-up study and chromosomal aberration test) prior to harvesting, colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation with colchicine, cell suspension was collected to pre-labeled tubes and centrifuged for 10 minutes at 1 500 to 1 800 rpm.
Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test material is considered to be clearly positive if, in any of the experimental conditions examined:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
b) The increase is concentration-related when evaluated with an appropriate trend test.
c) Any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test material is then considered to be able to induce chromosomal aberrations in cultured mammalian cells in this test system.
- Providing that all acceptability criteria are fulfilled, a test material will be considered clearly negative if, in all experimental conditions examined:
a) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
b) There is no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data.
The test material is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
An increase in the number of polyploid cells may indicate that the test material has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
An increase in the number of cells with endoreduplicated chromosomes may indicate that the test material has the potential to inhibit cell cycle progression.

Rationale for test conditions:

The in vitro chromosomal aberration test employ primary cell cultures derived from healthy human donor. The primary cell cultures of human whole blood are selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test systems.

Evaluation criteria:

Assay Acceptability Criteria
Study was accepted if;
- The concurrent vehicle control is within the 95 % control limits of the distribution of the laboratory’s historical negative/vehicle control database.
- Concurrent positive controls produced a statistically significant increase compared with the concurrent vehicle control and positive controls should induce responses that are compatible with those generated in the historical positive control database.
- Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analysable concentrations) were analysed.

Statistics:

Data (Percentage of cells with aberrations) was analysed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test material groups using ANOVA following Dunnett’s test and trend test at a 95 % level of confidence (p<0.05) and the statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: No change in pH was observed in any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.25, 0.5 and 1 µL/mL of test material.
- Water solubility: Test material was found miscible in DMSO at 200 µL/mL.
- Precipitation and time of the determination: Precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. After 24 hours of incubation, no precipitation was observed in any other concentrations tested up to 2 µL/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The concurrent vehicle control values for all treatment conditions were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.
Positive control, 10 µg/mL of Cyclophosphamide monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 12.70 % of aberrant cells which was statistically significant compared to the vehicle control (1.0 %). The reduction in mitotic index was 7.29 % when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 11.34 % of aberrant cells which was statistically significant to the vehicle control (1.33 %). The reduction in mitotic index observed was 7.07 % when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 9.67 % of aberrant cells which was statistically significant to the vehicle control (1.67 %). The reduction in mitotic index observed was 7.11 % when compared with the vehicle control for long term treatment.

- Results from cytotoxicity measurements: In the initial cytotoxicity test there was cytotoxicity in all the tested concentrations. Consequently a follow-up cytotoxicity test was performed at 0.0078, 0.0156, 0.0312, 0.625 and 0.125 µL/mL to assess the cytotoxicity and to select the appropriate test concentration for Chromosomal Aberration Test.
Treatment of cultures with the test material at concentrations 0.0078, 0.0156, 0.0312, 0.625 and 0.125 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) showed reduction in mitotic index and the observed values were 11.85, 14.69, 21.53, 30.21, 40.56 and 65.44 % respectively. In the absence of metabolic activation (short term treatment 3 to 6 hours) the obtained reduction in mitotic index was 10.17, 11.29, 18.28, 34.02, 41.75 and 64.52 % respectively. In the absence of metabolic activation (long term treatment 20 to 24 hours) the obtained reduction in mitotic index was 9.38, 12.80, 19.12, 26.13, 43.77 and 77.45 % respectively.

- Genotoxicity results
In the chromosomal aberration test, the cells were treated with the test material at the concentrations of 0.0312, 0.0625 and 0.125 µL/mL using DMSO as the vehicle in duplicates for short term (3 to 6 hours) both in the presence and absence of metabolic activation. Similarly, for long term (20 to 24 hours) in the absence of metabolic activation.
There was no statistically significant increase in the number of aberrant cells when compared with vehicle control at any of the concentration levels tested.
There was no increase in the number of aberrant cells when compared with the 95 % confidence level of laboratory’s historical vehicle control database at any of the concentrations tested and there was no concentration-related increase when evaluated with an appropriate trend test. There were no cells with endoreduplication chromosomes and no polyploidy cells were observed.
The reduction in MI observed at 0.125 µL/mL was 38.19 % in the presence of metabolic activation and 40.92 % in the absence of metabolic activation for short term treatments. Similarly, the reduction in MI observed at 0.125 µL/mL was 40.03 % in the absence of metabolic activation system for long term treatment.
The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.33 and 1.33 respectively. Similarly, the observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 1.33, 1.00 and 1.67 respectively. For both treatment conditions the observed mean percent aberrant cells for all concentrations of the test material were within the laboratory’s historical control data.
The observed mean percent aberrant cells at 0.0312, 0.0625 and 0.125 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.33, 1.33 and 1.00 respectively.

HISTORICAL CONTROL DATA
- Positive historical control data: In-house range of mean, standard deviation, margin of error and upper and lower bound with 95 % confidence level for Positive Control (Cyclophosphamide monohydrate, Mitomycin-C). Study period: November 2016 to October 2018
Average with S9 (10 µg/mL of CPA): 9.86 (SD 1.88) N = 27, margin of error 0.71. Upper bound 10.57, lower bound 9.15. 95 % confidence level 1.96. Max: 13.5, min: 6.70.
Average without S9 (3 - 6 h) (0.05 µg/mL of Mitomycin C): 9.86 (SD 2.02) N = 27, margin of error 0.76. Upper bound 10.62, lower bound 9.09. 95 % confidence level 1.96. Max: 12.67, min: 6.70.
Average without S9 (20 - 24 h) (0.05 µg/mL of Mitomycin C): 10.15 (SD 2.02) N = 27, margin of error 0.76. Upper bound 10.91, lower bound 9.38. 95 % confidence level 1.96. Max: 13.34, min: 6.70.

- Negative (solvent/vehicle) historical control data: In-house range of mean, standard deviation, margin of error and upper and lower bound with 95% confidence level for DMSO. Study period: November 2016 to October 2018
Average with S9: 0.67 (SD 0.31) N = 18, margin of error 0.14. Upper bound 0.81, lower bound 0.53. 95 % confidence level 1.96. Max: 1.67, min: 0.35.
Average without S9 (3 - 6 h): 0.79 (SD 0.39) N = 18, margin of error 0.18. Upper bound 0.97, lower bound 0.61. 95 % confidence level 1.96. Max: 2.0, min: 0.35.
Average without S9 (20 - 24 h): 0.72 (SD 0.33) N = 18, margin of error 0.15. Upper bound 0.87, lower bound 0.57. 95 % confidence level 1.96. Max: 1.33, min: 0.35.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material is considered as non-clastogenic up to the concentration of 0.0125 µL/mL both in the presence and absence of metabolic activation.

Executive summary:

The test material was evaluated for chromosomal aberrations in human lymphocytes according to OECD guideline 473 and in compliance with GLP.

The test material was found miscible in dimethyl sulphoxide at 200 µL/mL. A precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 24 hours of incubation, no precipitation was observed in any concentrations tested up to 2 µL/mL. No change in pH was observed in any of the concentration tested. Hence, 2 µL/mL was selected as the highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.25, 0.5, 1 and 2 µL/mL of test material.

In an initial cytotoxicity test, cytotoxicity was observed at all tested concentrations. Consequently, a follow-up cytotoxicity test was performed at 0.0078, 0.0156, 0.0312, 0.0625, 0.125 and 0.25 µL/mL to assess the cytotoxicity and to select the appropriate test concentration for Chromosomal Aberration Test.

In a follow-up cytotoxicity test, the cultures were treated with the test material at the concentrations of 0.0078, 0.0156, 0.0312, 0.0625, 0.125 and 0.25 µL/mL for short and long term treatment. The percentage reduction in Mitotic Index was in the range of 10.17 to 77.45 at 0.25, 0.125, 0.0625, 0.0312, 0.0156 and 0.0078 µL/mL. As the percentage reduction in MI was not more than 45 ± 5 % at 0.125 µL/mL, this was selected as the highest concentration for the chromosomal aberration test. Other concentrations selected were 0.0625 and 0.0312 µL/mL.

In the chromosomal aberration test, the cells were treated with the test material at the concentrations of 0.0039, 0.0078 and 0.0156 µL /mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mytomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, the cells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

There was no statistically significant increase in the number of aberrant cells in test material treated cultures when compared with vehicle control and there was no concentration-related increase when evaluated with an appropriate trend test. The reduction in MI observed at 0.125 µL/mL was 38.19 % in the presence of metabolic activation and 40.92 % in the absence of metabolic activation for short term treatments. Similarly, the reduction in MI observed at 0.125 µL/mL was 40.03 % in the absence of metabolic activation system for long term treatment.

The observed mean percent aberrant cells at 0.125, 0.0625 and 0.0312 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.0, 1.33 and 1.33 respectively, and fell within the 95 % confidence level of the laboratory’s historical control data. Similarly, the observed mean percent aberrated cells at 0.125, 0.0625 and 0.0312 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 1.33, 1.00 and 1.67 respectively, and fell within the 95 % confidence level of the laboratory’s historical control data.

The observed mean percent aberrated cells at 0.125, 0.0625 and 0.0312 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.33, 1.33 and 1.00 respectively, and fell within the 95 % confidence level of the laboratory’s historical control data.

The concurrent vehicle control values were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database. The cultures treated with positive controls for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation induced were 12.70 %, 11.34 % and 9.67 % of aberrant cells respectively, which was statistically significant compared with the respective vehicle control. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. 

Under the conditions of the study, the test material is considered as non-clastogenic up to the concentration of 0.0125 µL /mL both in the presence and absence of metabolic activation.