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Genetic toxicity in vitro

Description of key information

Negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions of this study.

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

Under the conditions of the study the test material is considered non-mutagenic in the gene mutation study in mammalian cells at and up to the concentration of 0.0625 µL/mL both in the presence and absence of metabolic activation.

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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
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
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.
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.
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.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2018 to 29 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
The derivative of the CHO-K1, CHO AA8 Cells were used as the test system as recommended in the guideline OECD 476.
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The derivative of the CHO-K1, CHO AA8 cells, Batch No. 5000062 procured from American Type Culture Collection (ATCC) was used for the test.
- Suitability of cells: The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting in vitro mammalian gene mutation test.

MEDIA USED
- Type and composition of media: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10 % Foetal Bovine Serum (FBS) and antibiotics (1 % penicillin and streptomycin) were used.
The pH of the culture medium was 7.34 to 7.36. The medium was stored at 2 to 8 ºC until use and thawed at room temperature before use.
Additional strain / cell type characteristics:
other: None specified
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: One mL of S9 homogenate was thawed immediately before use and mixed with 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 7.24 for the initial cytotoxicity test and pH 7.28 for the gene mutation test respectively. The S9 homogenate was prepared and stored in the test facility at -80 ± 10 ºC until use.
- Quality controls of S9: The batch of S9 homogenate was assessed for sterility, protein content (Modified Lowry assay, Sword and Thomson, 1980) and for its ability to metabolise the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.
Test concentrations with justification for top dose:
At a concentration of 0.0625 µL/mL the Relative Survival was in the range 10 to 20 %. Therefore 0.0625 µL/mL was selected as the highest concentration for testing in gene mutation test I.
Four concentrations i.e. 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL were selected for gene mutation test I, based on the initial cytotoxicity test.
In order to assess the reproducibility of the results the same concentrations were tested in gene mutation test II.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the maximum concentration or workable suspension of the test material in the vehicle compatible with the test system up to the maximum concentration of 200 µL/mL. Dimethyl sulphoxide was used to prepare the stock solution and dilutions of the test material. The test material was dissolved separately in a small amount of dimethyl sulphoxide. A solubility test was conducted in distilled water and dimethyl sulphoxide.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
PREPARATION OF CULTURES
A frozen cryovial of cells was thawed immediately in the water bath. Cells were transferred in to a sterile flask with culture medium containing 10 % FBS with antibiotics (1 % penicillin and streptomycin) and incubated at 37 ± 1 °C and 5 ± 1 % CO2 for 3 days. The cell lines were trypsinized and the trypsinized cultures were sub-cultured before use in the experiment. Approximately 2×10^6 cells per culture flask (initial cytotoxicity test and gene mutation test) were seeded using culture medium with 10 % FBS with antibiotics (1 % penicillin and streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the adjusted Cloning Efficiency. The flasks were incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 24 hours and 15 minutes (initial cytotoxicity test) to 25 hours (gene mutation test). Cells free of mycoplasma were used for the experiment.
The cultures were cleansed of pre-existing mutant cells by culturing in HAT Medium and then returned to normal growth medium.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Approximately 2×10^6 cells per culture flask (initial cytotoxicity test and gene mutation test) were seeded using culture medium with 10 % FBS with antibiotics (1 % penicillin and streptomycin).
- Test substance added in medium: In suspension.

FOR GENE MUTATION:
Each treatment group was maintained with four plate cultures. Cells were exposed to the test material for 3 hours and 15 minutes (gene mutation test I) and 3 hours and 40 minutes (gene mutation test II) for both with exogenous metabolic activation and without exogenous metabolic activation respectively in the gene mutation tests at 37 ± 1 °C with 5 ± 1 % CO2.
Four plate treatments were pooled into a pre-labelled tube and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained.
- Expression time: The replicate cultures were sub-cultured in duplicates at a density of 1×10^6 cells/culture flask. Cells were incubated at 37 ± 1 °C with 5 ± 1 % CO2, followed by sub-culturing with an interval of 2 to 3 days for the remaining 9 days of expression period.
- Selection time: Following the 9 day expression period, each replicate treatment culture was pooled and sub-cultured in quintuplicates at a density of 4×10^5 cells per group with culture media containing 10 µM of 6-Thioguanine and 200 cells/ dish in triplicate without 6-Thioguanine for determination of cloning efficiency. Dishes were incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 9 days. After the incubation period, medium from each dish was aspirated and stained with 5 % Giemsa stain and the number of colonies formed were counted manually.
- Method used: Agar

Mutant Frequency of each treatment was calculated using the following formula:
Mutant Frequency (MF) = (cloning efficiency of mutant colonies in selective medium / cloning efficiency in non-selective medium).
Cloning efficiency = Number of colonies / number of cells plated.
MF is expressed as mutants per 2 x 10^6 clonable cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS)
- Any supplementary information relevant to cytotoxicity: Based on the results of solubility, pH and precipitation test an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test. Five concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL of the test material were tested in an initial cytotoxicity test.
For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 µL of vehicle/different concentrations of test material was added to four plates per cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test material for 4 hours at 37 ± 1 °C with 5 ± 1 % CO2.
For tests without exogenous metabolic activation, a volume of 100 µL of vehicle/different concentrations of test material was added to four plates per culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test material for 4 hours at 37 ± 1 °C with 5 ± 1 % CO2.
Post the incubation period (Set 1 and 2), medium from each flask was aspirated and the monolayer was washed with DPBS. Cells were trypsinized. Trypsinization was stopped by adding culture media followed by collecting the media with cells.
Four plate treatments were collected in pre-labelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained.
Each treatment replicate was plated in triplicate with a cell concentration of 200 cells/ 5 mL media in cell culture 25 cm^2 dishes and incubated at 37 ± 1 °C with 5 ± 1 % CO2 for 7 - 8 days.
After the incubation period, medium from each culture flask was aspirated and stained with 5 % Giemsa stain. The number of colonies formed was counted manually.
The cytotoxicity level was determined using the following formulae:

Adjusted cloning efficiency (ACE) = CE x (no. of cells at the end of treatment / No. of cells at the beginning of the treatment)

Relative survival (RS) = (ACE (treatetd) / ACE (vehicle control)) x 100

Cloning efficiency (CE) is the percentage of cells plated at a low density that are able to grow into a colony that can be counted.

PRECIPITATION AND PH TEST
A precipitation test was conducted up to 2 µL/mL to determine the ability of the test material to cause precipitation in the medium. A quantity of 100 µL of test material was mixed with 10 mL of culture media and incubated at 37 ± 1 ºC with 5 ± 1 % CO2 for 4 hours. Post incubation, results were recorded for change in pH and signs of precipitation.

INTERPRETATION OF RESULTS
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- The increase is concentration-related when evaluated with an appropriate trend test,
- Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
- A test chemical is considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- There is no concentration-related increase when evaluated with an appropriate trend test,
- All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above, performing a repeat experiment possibly using modified experimental conditions will be considered in consultation with the sponsor.
Evaluation criteria:
Acceptance of a test is based on the following criteria:
- The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative/vehicle control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
- The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.
Statistics:
Data of mutant frequencies were analysed using SPSS Software version 22 for differences among vehicle control, treatment and positive control groups and linear trend using ANOVA following Dunnett’s test at a 95 % level of significance. The statistical significances are designated by the superscripts as given below:
** Statistically significant (p<0.05) change than the vehicle control group.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test material was miscible in DMSO at 200 µL/mL and pH tested at concentrations upto 2 µL/mL was comparable to the vehicle control. Based on these results, 2 µL/mL was selected as the highest concentration for the initial cytotoxicity test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Gene mutation test I: The positive control, 3 µg/mL of Benzo(a)pyrene, in the presence of metabolic activation, resulted in a RS value of 72.92 % and mutant frequency of 271.25 per 2×10^6 cells which was statistically significant when compared with the vehicle control.
The positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, in the absence of metabolic activation, resulted in a RS value of 73.63 % and mutant frequency of 263.75 per 2×10^6 cells and was statistically significant when compared with that of vehicle control.
Gene mutation test II: The positive control, 3 µg/mL of Benzo (a) pyrene, in the presence of metabolic activation, resulted in a RS value of 86.73 % and mutant frequency of 270.24 per 2×10^6 cells which was statistically significant when compared with the vehicle control.
The positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, in the absence of metabolic activation, resulted in a RS value of 78.76 % and mutant frequency of 259.30 per 2×10^6 cells and was statistically significant when compared with that of vehicle control.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: In the initial cytotoxicity test, excessive cytotoxicity (>10 % RS) was observed at concentrations of 0.125 µL/mL and above in both presence of metabolic activation and absence of metabolic activation. Following treatment at 0.0625 µL/mL cytotoxicity was 20.22 % RS in the presence of S9 and 22.73 % in the absence of S9. The results of the initial cytotoxicity test indicated that an acceptable degree of cytotoxicity was induced only at 0.0625 µL/mL (10 to 20 % RS, when compared with the respective vehicle control) both in the presence and absence of metabolic activation.
- Genotoxicity results:
Gene mutation test I: In the gene mutation test, the cells were treated with test material at the concentrations of 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL using DMSO as the vehicle in four plate cultures both in the presence of metabolic activation and absence of metabolic activation.
The test material resulted in mutant frequencies of 25.30 to 30.38 per 2×10^6 cells in the presence of metabolic activation with 25.88 per 2×10^6 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 28.24 to 32.50 per 2×10^6 cells were observed with 26.44 per 2×10^6 cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the tested concentrations and there was no concentration-related increase when evaluated with an appropriate trend test. However, the majority of treated cultures exceed the laboratory’s historical control data range.
There was no evidence of excessive cytotoxicity (<10 % RS) at any of the concentrations in both the presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 22.92 to 73.96 % and in the absence of metabolic activation the RS values ranged from 23.08 to 74.73 % respectively.
Gene mutation test II: There was no evidence of excessive cytotoxicity (<10 % RS) at any of the concentrations in both presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 33.63 to 89.38 % and in the absence of metabolic activation the RS values ranged from 38.05 to 82.30 % respectively.
The test material resulted in mutant frequencies of 25.00 to 25.27 per 2×10^6 cells in the presence of metabolic activation with 23.16 per 2×10^6 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 25.27 to 26.67 per 2×10^6 cells were observed with 24.47 per 2×10^6 cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the tested concentrations and there was no concentration-related increase when evaluated with an appropriate trend test.
These results confirm that the test material does not induce gene mutations in this assay system and that the outside of historical control responses observed in gene mutation test I are not reproducible and consequently of no biological relevance.

HISTORICAL CONTROL DATA
- Positive historical control data: Benzo(a)pyrene study period July 2017 to November 2018.
Mean data of mutant frequency/ 2 x 10^6 cells with metabolic activation (3 to 6 h): 261.94 (SD 27.28), margin of error 17.82; upper bound 279.76, lower bound 244.12.
- Positive historical control data: 4 Nitroquinoline N-oxide study period July 2017 to November 2018.
Mean data of mutant frequency/ 2 x 10^6 cells without metabolic activation (3 to 6 h): 264.60 (SD 18.52), margin of error 12.10; upper bound 276.70, lower bound 252.50.
- Negative historical control data: DMSO vehicle control study period July 2017 to November 2018.
Mean data of mutant frequency/ 2 x 10^6 cells with metabolic activation (3 to 6 h): 24.51 (SD 2.81), margin of error 1.95; upper bound 26.46, lower bound 22.56.
Mean data of mutant frequency/ 2 x 10^6 cells without metabolic activation (3 to 6 h): 25.43 (SD 1.89), margin of error 1.31; upper bound 26.74, lower bound 24.12.
Conclusions:
Under the conditions of the study the test material is considered non-mutagenic at and up to the concentration of 0.0625 µL/mL both in the presence and absence of metabolic activation.
Executive summary:

The test material was evaluated for gene mutation in CHO AA8 cells, according to OECD guideline 476 and in compliance with GLP.

The test material was found to be miscible in DMSO at 200 µL/mL. There was no change in pH and no precipitate following addition of the test material at and up to 2 µL/mL in culture medium. Based on the results of solubility, pH and precipitation test an initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL using DMSO as a vehicle in 4 plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the test material was cytotoxic to CHO AA8 cells at concentrations of 0.125 µL/mL and above. Relative Survival of the treated CHO AA8 cells at 0.0625 µL/mL was above 20 % relative survival (20.22 % (+S9) and 22.73 % (-S9) in the initial cytotoxicity test and 22.92 % (+S9) and 23.08 % (-S9) in the gene mutation test) when compared with the respective vehicle control, both in the presence and absence of metabolic activation.

The gene mutation test I (3 hours and 15 minutes) and II (3 hours and 40 minutes) were conducted at concentrations of 0.0078, 0.0156, 0.0312, and 0.0625 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation.

Benzo(a)pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation tests.

Cytotoxicity was assessed by determining the adjusted Cloning Efficiency and Relative Survival in the test.

In gene mutation test I the mutant frequencies obtained at concentrations of 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL were not inside the distribution of the historical vehicle control data. Consequently, as the response was not clearly negative or clearly positive as described in the study plan, a second experiment (gene mutation test II) was conducted in consultation with the sponsor.

There was no statistically significant increase in the number of mutant colonies and there was no concentration-related increase when evaluated with an appropriate trend test at any of the concentration tested in the both gene mutation test I and II. In gene mutation test I mutant colonies in the test material were outside the historical control data and in gene mutation test II mutant colonies were within the historical control data, historical control responses observed in gene mutation test I are not reproducible and consequently of no biological relevance.

The concurrent vehicle control values for both experiments were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.

Positive controls resulted in mutant frequencies, which were statistically significant when compared with the respective vehicle control.

Under the conditions of the study the test material is considered non-mutagenic at and up to the concentration of 0.0625 µL/mL both in the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Principles of method if other than guideline:
Method: other: comparable to guidelines established by OECD, USEPA, and USFDA
All tester strains were checked for the presence of the appropriate genetic markers. The S. typhimurium strains were obtained from Dr. Bruce Ames, University of California - Berkeley. The E. coli strain was obtained from Dr. M.H.L. Green, M.R.C. Cell Mutation Unit, University of Sussex, Falmer, England. For tests with metabolic activation, a reaction mixture was prepared with 8 mM of MgCl2, 33 mM KCl, 4 mM NADP, 5 mM glucose-6-phosphate, 100 mM Na2HPO4 (pH 7.4), and 6% (v/v) S9 Homogenate fraction. The S9 Homogenate was prepared at Pharmakon Research from Aroclor 1254-induced male Sprague-Dawley rat liver homogenate, according to the procedures of Maron and Ames (1983). The concentration of liver homogenate in the metabolic activation mixture was optimized for each lot of S9 prepared using 3 positive control articles (2-acetylaminofluorene, 2-anthramine, and benzo[a]pyrene). [Maron, D.M. and B.N. Ames. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215.] All required dilutions of the test substance were made with dimethyl sulfoxide (DMSO). Dilutions were prepared the day of the test and used immediately after preparation. A preliminary toxicity screen was conducted, using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100, and E. coli WP2 uvrA (treated with the test substance at doses of 50, 167, 500, 1670, and 5000 ug/plate), and a DMSO solvent control were prepared without metabolic activation. Following a 48-hour incubation at 37 deg. C, the background lawn and spontaneous revertants were scored for normal, inhibited, or no growth. Primary test cultures were prepared from working stock cultures in 25 mL of Oxoid nutrient broth #2, incubated for ~8 hours at 37 deg. C, and diluted 1:4 in de-ionized water. Optical densities were determined at 650 nm and cultures with optical densities of 0.4-0.6 (~1-2 x 10E9 cells/mL) were used for this study. The test substance was evaluated for mutagenicity using both liquid pre-incubation and plate incorporation treatment conditions. Triplicate cultures of all six test strains were prepared and evaluated at doses of 16.7, 50, 167, 500, 1670, and 5000 ug/plate, both with and without metabolic activation (S9). Positive and negative control articles (with and without activation) were prepared in triplicate for the primary mutagenicity testing:
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
selected histidine loci
tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
exogenous metabolic activation system (S9)
Test concentrations with justification for top dose:
16.7, 50, 167, 500, 1670, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent giving the best solubility or suspension was selected.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
all tester strains plated with DMSO ±S9
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: 2-aminofluorene; 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); liquid preincubation;

DURATION
- Preincubation period: approximately 30 minutes for those undergoing liquid pre-incubation
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate cultures


Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan- independent revertants, with at least one dose level inducing a revertant frequency that is two-fold the concurrent negative control value. Statistical analysis were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the tetst article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants. Inhibited growth was characterized by the absence of a confluent bacterial lawn nd /or the presence of pindot colonies.
Statistics:
Statistical analyses were conducted using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants, with at least one dose level inducing a revertant frequency that was two-fold the solvent control value. An equivocal result was declared if the test substance did not induce a statistically significant, dose-dependent increase in revertant frequency, but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value. A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants. Statistical analyses were only conducted when a 50% increase in revertant frequency (relative to the concurrent negative controls) was observed.
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none noted
- Water solubility: DMSO- solvent

RANGE-FINDING/SCREENING STUDIES:
Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 urvA were treated with the test substance at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control in the absence of S9 activation. Results of the pre-screen indicated that the test substance produced inhibited growth (characterised by the absence of a confluent bacterial lawn and /or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre-incubation conditions. In addition, the test substance was found to be incompletely soluble at doses ≥500 µg/plate.
Preliminary test results showed that the test substance produced inhibited growth in both Salmonella tester strains at doses ≥500 µg/plate, under liquid pre-incubation conditions. Primary test results showed that, except for strain WP2 uvrA, inhibited growth was again observed for all test strain/S9/treatment combinations at the 3 highest dose levels (i.e., 500, 1670 and 5000 µg/plate). In addition, the test substance again was seen to be incompletely soluble at levels ≥500 µg/plate. Statistically significant increases in revertant frequencies (to ~1.6 -2.0 fold control values) were observed in strains TA1537 and TA1535 at a dose level of 16.7 µg/plate, without S9, under liquid pre-incubation and plate incorporation conditions, respectively. However, these increases were not determined to be dose dependent and the observed revertant frequencies approximated historical negative control values. Revertant frequencies for all other dose/test strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values (in both assays) were within acceptable ranges. Therefore, the slight increases observed in strains TA1535 and TA1537 were considered statistical aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical data were provided for positive and negative controls. Six dose levels were evaluated in case of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutation assay. After a 48-hour incubation at 37 °C, all plates were scored for bacterial growth. Solvent and positive controls were scored first, and test substance-treated cultures were scored only if the average negative control values were within historical ranges.
Conclusions:
Negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions of this study.
Executive summary:

The test article, methyltin tris(2­ethylhexylthioglycolate), was evaluated in the Ames/Salmonella­E. coli Reverse Mutation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102), and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9).

Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre­incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 uvrA were treated at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control, in the absence of S9. Results of the pre-screen indicated the test material produced inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre­incubation conditions. In addition, the test article was found to be incompletely soluble at doses ≥500 µg/plate. 

The test article next was evaluated for mutagenicity using both treatment conditions. Based upon the results of the pre-screen, the test material was evaluated in triplicate cultures in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9. Six doses were evaluated in the event of unacceptable toxicity and/or insolubility at the highest dose levels in the mutation assay. The S9 mixture included 6 % (v/v) Aroclor 1254­induced male Sprague­Dawley rat liver homogenate with the appropriate buffer and cofactors.

Except for strain WP2 uvrA, inhibited growth again was observed for all tester strain/S9/treatment combinations at the highest 1­3 doses evaluated. In addition, the test article again was found to be incompletely soluble at doses ≥500 µg/plate. Statistically significant increases in revertant frequencies, to approximately 1.6­ to 2.0­fold control values, were observed in strains TA1537 and TA1535 at a dose of 16.7 µg/plate without S9 under liquid pre­incubation and plate incorporation conditions, respectively. However, these increases were not dose dependent, and the observed revertant frequencies approximated historical negative control values. In addition, revertant frequencies for all other dose/tester strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values in both assays were within acceptable ranges. Thus, the slight increases observed in strains TA1535 and TA1537 are considered to be statistical aberrations due to random fluctuation of the spontaneous revert frequencies.

Therefore, the results for the test article, methyltin tris(2­ethylhexylthio­glycolate), were negative in the Ames/Salmonella­E. coli Reverse Mutation Assay, using liquid pre­incubation and plate incorporation treatments, under the conditions of the study according to the criteria of the test protocol.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test: Stankowski (1996)

The test article, methyltin tris(2­ethylhexylthioglycolate), was evaluated in the Ames/Salmonella­E. coli Reverse Mutation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102), and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre­incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 uvrA were treated at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control, in the absence of S9. Results of the pre-screen indicated the test material produced inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre­incubation conditions. In addition, the test article was found to be incompletely soluble at doses ≥500 µg/plate. 

The test article next was evaluated for mutagenicity using both treatment conditions. Based upon the results of the pre-screen, the test material was evaluated in triplicate cultures in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9. Six doses were evaluated in the event of unacceptable toxicity and/or insolubility at the highest dose levels in the mutation assay. The S9 mixture included 6 % (v/v) Aroclor 1254­induced male Sprague­Dawley rat liver homogenate with the appropriate buffer and cofactors.

Except for strain WP2 uvrA, inhibited growth again was observed for all tester strain/S9/treatment combinations at the highest 1­3 doses evaluated. In addition, the test article again was found to be incompletely soluble at doses ≥500 µg/plate. Statistically significant increases in revertant frequencies, to approximately 1.6­ to 2.0­fold control values, were observed in strains TA1537 and TA1535 at a dose of 16.7 µg/plate without S9 under liquid pre­incubation and plate incorporation conditions, respectively. However, these increases were not dose dependent, and the observed revertant frequencies approximated historical negative control values. In addition, revertant frequencies for all other dose/tester strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values in both assays were within acceptable ranges. Thus, the slight increases observed in strains TA1535 and TA1537 are considered to be statistical aberrations due to random fluctuation of the spontaneous revert frequencies.

Therefore, the results for the test article, methyltin tris(2­ethylhexylthio­glycolate), were negative in the Ames/Salmonella­E. coli Reverse Mutation Assay, using liquid pre­incubation and plate incorporation treatments, under the conditions of the study according to the criteria of the test protocol.

Chromosome Aberration Test: Jagadeesh (2019)

The test material was evaluated for chromosomal aberrations in human lymphocytes according to OECD guideline 473 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 withthe 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/mLwas 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 at0.125 µL/mL was 40.03 % in the absence of metabolic activation systemfor 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 aberrant 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 aberrant 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.

Theconcurrent 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 controlsfor 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.

In Vitro Gene Mutation Study in Mammalian Cells: Jagadeesh (2019)

The test material was evaluated for gene mutation in CHO AA8 cells, according to OECD guideline 476 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was found to be miscible in DMSO at 200 µL/mL. There was no change in pH and no precipitate following addition of the test material at and up to 2 µL/mL in culture medium. Based on the results of solubility, pH and precipitation test an initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.50, 1 and 2 µL/mL using DMSO as a vehicle in 4 plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the test material was cytotoxic to CHO AA8 cells at concentrations of 0.125 µL/mL and above. Relative Survival of the treated CHO AA8 cells at 0.0625 µL/mL was above 20 % relative survival (20.22 % (+ S9) and 22.73 % (-S9) in the initial cytotoxicity test and 22.92 % (+S9) and 23.08 % (-S9) in the gene mutation test) when compared with the respective vehicle control, both in the presence and absence of metabolic activation.

The gene mutation test I (3 hours and 15 minutes) and II (3 hours and 40 minutes) were conducted at concentrations of 0.0078, 0.0156, 0.0312, and 0.0625 µL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation.

Benzo(a)pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation tests.

Cytotoxicity was assessed by determining the adjusted Cloning Efficiency and Relative Survival in the test.

In gene mutation test I the mutant frequencies obtained at concentrations of 0.0078, 0.0156, 0.0312 and 0.0625 µL/mL were not inside the distribution of the historical vehicle control data. Consequently, as the response was not clearly negative or clearly positive as described in the study plan, a second experiment (gene mutation test II) was conducted in consultation with the sponsor.

There was no statistically significant increase in the number of mutant colonies and there was no concentration-related increase when evaluated with an appropriate trend test at any of the concentration tested in the both gene mutation test I and II. In gene mutation test I mutant colonies in the test material were outside the historical control data and in gene mutation test II mutant colonies were within the historical control data, historical control responses observed in gene mutation test I are not reproducible and consequently of no biological relevance.

The concurrent vehicle control values for both experiments were within the 95 % control limits of the distribution of the laboratory’s historical vehicle control database.

Positive controls resulted in mutant frequencies, which were statistically significant when compared with the respective vehicle control.

Under the conditions of the study the test material is considered non-mutagenic at and up to the concentration of 0.0625 µL/mL both in the presence and absence of metabolic activation.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to mutagenicity.