Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay: Negative (with and without metabolic activation in Salmonella typhimurium and e. coli strains)

Gene mutation assay: Negative (with and without metabolic activation in mouse lymphoma L5178Y TK+/- cells)

Chromosome Aberration: Negeative (with and without metabolic activation in Chinese hamster V79 lung cells)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2006 - 11 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB- (salmonella typhimurium)
WP2urvA- (Escherichia coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mixture
Test concentrations with justification for top dose:
In the prelimary toxicity test the concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Mutation Test experiment 1 & 2, five concentrations of the test material (50, 150, 500m 1500 and 500 µg/plate) were assayed in triplicate agains eachtester strain.
Vehicle / solvent:
The test material was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully soluble in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80µg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2µg/plate for TA98
Details on test system and experimental conditions:
S9-Mix and Agar
The S9-mix was prepared immediately before using sterilised co0factors and maintained on ice for the duration of the test
S9 5.0 mL
1.5 M KCL/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADPH 2.0 mL
0. M NADH 2.0 mL
0.2 M Sodium phsopahate buffer (pH 7.4) 25.0 mL
Sterile distilled water 12.5 mL
A 0.5 mL aliquot of S9-mix and 2mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix .
Top agag was prepared using 0.% Difco Bacto agar (lot no. 2102667 01/10) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100mL of top agar.

Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 j.lg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate ). Ten concentrations of the test material formulation and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 3 7°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).
Evaluation criteria:
There are several criteria for determining a positive results, such as a dose-related increase in revertant frequency over the dose range tested and/or reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS(%) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale particulate precipitate was observed at and above 1500 µg/plate this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or withoutmetabolic activation.

All of the positive control chemical used in the test induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 The test material was non-toxic to the strains of bacterial used (TA100 and WP2uvr A-). The test material formulation S9-Mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

 -

TA100

104

106

104

91

86

99

100

80

98

91P

108P

TA100

96

76

64

78

84

80

66

85

75

91P

101P

 -

Wp2uvrA-

23

20

18

24

22

16

21

20

19

24P

22P

 +

Wp2uvrA-

31

19

29

36

29

21

31

27

26

34P

31P

P - Precipitate

Conclusions:
Interpretation of results (migrated information):
negative

Test material was considered to be non-mutagenic under the conditions of this study.
Executive summary:

The aim of the study was to assess the mutagenic potential of the test material Propanoic acid, 3-(dodecylthio)-,2,2-bis[[3-dodecylthio)-1-oxopropoxy]methyl]-1,3-propanediyl ester using a bacterial/microsome test system.

The study was based on the in vitro technique Ames test which mutagenic activity was assess by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of E-coli to various concentrations of the test material. This was done in accordance to the following guidelines:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

EU Method B13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria).

The test material was considered to be non-mutagenic under the conditions of the study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 July 2017 to 08 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity- In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
thymidine kinase (tk) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 1661603 / 60797977
Supplier: American Type Culture Collection
(Manassas,Virginia, USA)
Date of receipt: 22 January 2004 / 27 February 2014
Date of working lot: 21 January 2016 (MP13) / 17 May 2017 (MP14)*
*Note: MP13 was used in the preliminary experiment, MP14 was used in the main tests.
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the main tests (cells were subcultured 7 times when used in the preliminary test).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,
Assay 1, 3-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,
Assay 2, 24-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL.
Vehicle / solvent:
Based on the available information (trial formulations performed at the Test Facility) 1% (w/v) aqueous methyl cellulose solution (abbreviations of 1% methyl cellulose or 1% MC were used in the report text) was selected for vehicle of the study.

Name: Methyl cellulose
Supplier: HungaroPharma
Lot No.: 5115851
Appearance: White to off white powder
Expiry date: 27 November 2018
Storage conditions: Room temperature
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
Preliminary Toxicity Test
A preliminary toxicity test was performed to select dose levels for the main assays.
During the preliminary test, a 3-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity.
The procedures were performed as described in Main Mutation Assays; however, the test used single cultures and positive controls were not included. The highest test concentration in the preliminary test was 2000 μg/mL (the recommended maximum concentration).
Following treatments, cell concentrations were determined using a haemocytometer.
Visual examination for any kind of insolubility in the final culture medium was conducted at the beginning and end of treatment. Measurement of pH and osmolality was performed at the end of the treatment period. Then cells were transferred for the expression period for two extra days and repeated cell counting was performed.

Main Mutation Assays
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
A suitable volume (0.2 mL) of RPMI-5 medium, vehicle (solvent), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37 °C ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer.
Where sufficient cells survived, cell density was adjusted to a concentration of 2x105 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.

Plating for Survival
Cultures of cell density 2x105 cells/mL, were further diluted to 8 cells/mL.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days.
During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2x105 cells/mL (whenever possible) and transferred to flasks for further growth (total volume of 30 mL per flask).
On completion of the expression period, six test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1x104 cells/mL with RPMI-20 for plating for a viability test.
Samples from these cultures were diluted to 8 cells/mL.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for -trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1x104 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2x103 cells per well.
Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.
Rationale for test conditions:
In accordance with test guidelines
Evaluation criteria:
The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.
The negative (vehicle) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY EXPERIMENT
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment using 1% methyl cellulose as vehicle was 2000 μg/mL (the recommended maximum concentration).
No cytotoxicity, but insolubility was detected in the preliminary experiments.
Therefore, concentrations the recommended maximum concentration was selected for the highest concentration of the main experiments, spacing was selected in such a way that cover the range from insolubility to no or little insolubility according to the instructions of the relevant OECD guideline. Six concentrations were selected for the main experiments in each assay.

MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (+S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel sub-cultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks (12 days) in culturing plates with and without selective agent (TFT) for determination of mutations and viability.
Assay 1
In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL in both experiments.
In Assay 1, no large changes in pH or osmolality were detected. Precipitate / minimal amount of precipitate was observed in the final treatment medium at the end of the treatment at all test item concentrations.
In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of
97%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this experiment was considered as being negative.
In the absence of S9-mix (3-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 67%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was also considered as being negative.
Assay 2
In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL in both experiments.
In Assay 2, no large changes in pH or osmolality were detected. Precipitate / minimal amount of precipitate was observed in the final treatment medium at the end of the treatment at all test item concentrations.
In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed as are shown in Table 3 of Appendix 5. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 93%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this result confirmed the negative result observed in the first main test with metabolic activation.
In the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of
123%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result of the first main test without metabolic activation.
Other minor increases in the mutation frequency were observed sporadically in Assays 1 and 2; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they were considered as biologically not relevant increases, just showing the biological variability of the test system.

VALIDITY OF THE MUTATION ASSAYS
Untreated, negative (vehicle) and positive controls were run concurrently in the study.
The spontaneous mutation frequency of the negative (vehicle) and untreated controls were in the recommended range in all cases.
The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD criteria.
The plating efficiencies for the negative (vehicle) control of the test item and untreated control samples at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assay.
The number of test concentrations evaluated was six in each case, which met the acceptance criteria of at least four evaluated concentrations.
The tested concentration range in the study was considered to be adequate as concentrations up to the recommended maximum concentration were examined in the study, furthermore the selected range covered concentrations from insolubility to no or little insolubility. Lower test concentrations were spaced by a factor of three.
Suspension growth value of the untreated and negative (vehicle) control were within the recommended range in all cases.
The overall study was considered to be valid.

Results of Preliminary Toxicity Test

(3-hour treatment in the presence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment Day 0

Total cell number (Relative Survival*, %) on Day 1

Total cell number (Relative Survival*, %) on Day 2

Observations at the beginning / after treatment

Untreated control

7.15E+06 (92)

2.10E+07 (117)

2.00E+07 (90)

normal / normal (pH: 7.0, osmolality: 298 mmol/kg)

Negative (vehicle) control

7.75E+06 (100)

1.80E+07 (100)

2.22E+07 (100)

normal / normal (pH: 7.0, osmolality: 294 mmol/kg)

2000 μg/mL

6.75E+06 (87)

2.03E+07 (113)

2.16E+07 (97)

precipitate / precipitate (pH: 7.0, osmolality: 297 mmol/kg)

1000 μg/mL

6.15E+06 (79)

2.10E+07 (117)

2.26E+07 (102)

precipitate / precipitate (pH: 7.0, osmolality: 298 mmol/kg)

500 μg/mL

6.75E+06 (87)

2.01E+07 (112)

2.02E+07 (91)

precipitate / precipitate (pH: 7.0, osmolality: 294 mmol/kg)

250 μg/mL

6.95E+06 (90)

2.04E+07 (113)

1.94E+07 (87)

precipitate / precipitate (pH: 7.0, osmolality: 295 mmol/kg)

125 μg/mL

7.15E+06 (92)

2.07E+07 (115)

2.14E+07 (96)

precipitate / precipitate (pH: 7.0, osmolality: 294 mmol/kg)

62.5 μg/mL

7.10E+06 (92)

2.10E+07 (117)

2.10E+07 (95)

precipitate / precipitate (pH: 7.0, osmolality: 296 mmol/kg)

31.25 μg/mL

6.25E+06 (81)

2.09E+07 (116)

2.42E+07 (109)

precipitate# / precipitate# (pH: 7.0, osmolality: 297 mmol/kg)

15.65 μg/mL

6.70E+06 (86)

1.77E+07 (98)

2.06E+07 (93)

precipitate# / precipitate# (pH: 7.0, osmolality: 294 mmol/kg)

7.813 μg/mL

7.10E+06 (92)

1.92E+07 (107)

2.04E+07 (92)

precipitate# / precipitate# (pH: 7.0, osmolality: 297 mmol/kg)

3.906 μg/mL

6.60E+06 (85)

2.22E+07 (123)

2.14E+07 (96)

precipitate# / precipitate# (pH: 7.0, osmolality: 294 mmol/kg)

*: compared to the relative negative (vehicle) control (1% methyl cellulose)        #: minimal amount

 

Results of Preliminary Toxicity Test

(3-hour treatment in the absence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment Day 0

Total cell number (Relative Survival*, %) on Day 1

Total cell number (Relative Survival*, %) on Day 2

Observations at the beginning / after treatment

Untreated control

1.15E+07 (104)

1.95E+07 (87)

2.16E+07 (95)

normal / normal (pH: 7.0, osmolality: 294 mmol/kg)

Negative (vehicle) control

1.11E+07 (100)

2.25E+07 (100)

2.28E+07 (100)

normal / normal (pH: 7.0, osmolality: 291 mmol/kg)

2000 μg/mL

1.19E+07 (107)

1.74E+07 (77)

2.28E+07 (100)

precipitate / precipitate (pH: 7.0, osmolality: 291 mmol/kg)

1000 μg/mL

1.04E+07 (94)

2.19E+07 (97)

2.66E+07 (117)

precipitate / precipitate (pH: 7.0, osmolality: 288 mmol/kg)

500 μg/mL

1.27E+07 (114)

2.12E+07 (94)

2.08E+07 (91)

precipitate / precipitate (pH: 7.0, osmolality: 289 mmol/kg)

250 μg/mL

9.30E+06 (84)

2.27E+07 (101)

2.14E+07 (94)

precipitate / precipitate (pH: 7.0, osmolality: 288 mmol/kg)

125 μg/mL

1.14E+07 (103)

2.01E+07 (89)

2.38E+07 (104)

precipitate / precipitate (pH: 7.0, osmolality: 289 mmol/kg)

62.5 μg/mL

9.30E+07 (84)

2.46E+07 (109)

2.02E+07 (89)

precipitate / precipitate (pH: 7.0, osmolality: 289 mmol/kg)

31.25 μg/mL

1.11E+07 (100)

2.04E+07 (91)

2.50E+07 (110)

precipitate# / precipitate# (pH: 7.0, osmolality: 297 mmol/kg)

15.65 μg/mL

9.55E+06 (86)

1.74E+07 (77)

2.30E+07 (101)

precipitate# / precipitate# (pH: 7.0, osmolality: 289 mmol/kg)

7.813 μg/mL

1.14E+07 (103)

1.68E+07 (75)

1.90E+07 (83)

precipitate# / precipitate# (pH: 7.0, osmolality: 289 mmol/kg)

3.906 μg/mL

9.30E+07 (86)

1.94E+07 (86)

1.96E+07 (86)

precipitate# / precipitate# (pH: 7.0, osmolality: 289 mmol/kg)

*: compared to the relative negative (vehicle) control (1% methyl cellulose)        #: minimal amount

 

Results of Preliminary Toxicity Test

(24-hour treatment in the absence of metabolic activation)

Test item (or solvent) concentration

Total cell number (Relative Survival*, %) after treatment Day 1

Total cell number (Relative Survival*, %) on Day 2

Total cell number (Relative Survival*, %) on Day 3

Observations at the beginning / after treatment

Untreated control

1.49E+07 (105)

1.95E+07 (92)

2.48E+07 (102)

normal / normal (pH: 7.0, osmolality: 290 mmol/kg)

Negative (vehicle) control

1.42E+07 (100)

2.12E+07 (100)

2.44E+07 (100)

normal / normal (pH: 7.0, osmolality: 289 mmol/kg)

2000 μg/mL

1.77E+07 (125)

2.19E+07 (104)

2.16E+07 (89)

precipitate / precipitate (pH: 7.0, osmolality: 289 mmol/kg)

1000 μg/mL

1.46E+07 (102)

2.37E+07 (112)

2.28E+07 (93)

precipitate / precipitate (pH: 7.0, osmolality: 290 mmol/kg)

500 μg/mL

1.68E+07 (118)

2.22E+07 (105)

2.02E+07 (83)

precipitate / precipitate (pH: 7.0, osmolality: 291 mmol/kg)

250 μg/mL

1.88E+07 (132)

2.36E+07 (111)

2.00E+07 (82)

precipitate / precipitate (pH: 7.0, osmolality: 289 mmol/kg)

125 μg/mL

1.53E+07 (107)

2.19E+07 (104)

2.16E+07 (89)

precipitate / precipitate (pH: 7.0, osmolality: 292 mmol/kg)

62.5 μg/Ml

1.53E+07 (108)

2.27E+07 (107)

2.02E+07 (83)

precipitate / precipitate (pH: 7.0, osmolality: 290 mmol/kg)

31.25 μg/mL

1.61E+07 (113)

2.07E+07 (98)

2.40E+07 (98)

precipitate# / precipitate# (pH: 7.0, osmolality: 291 mmol/kg)

15.65 μg/mL

1.46E+07 (103)

2.01E+07 (95)

2.32E+07 (95)

precipitate# / precipitate# (pH: 7.0, osmolality: 290 mmol/kg)

7.813 μg/mL

1.63E+07 (114)

2.31E+07 (109)

2.14E+07 (88)

precipitate# / precipitate# (pH: 7.0, osmolality: 289 mmol/kg)

3.906 μg/mL

1.39E+08 (98)

1.97E+07 (92)

2.16E+07 (89)

precipitate# / precipitate# (pH: 7.0, osmolality: 288 mmol/kg)

*: compared to the relative negative (vehicle) control (1% methyl cellulose)        #: minimal amount

 

Summary Tables of Survival Data

 

Survival Results Assay 1 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

Relative Survival§

(%RS)

Relative Total Growth

(%RTG)

+

3

A1

2000 μg/mL

64/384

1.120

108

97

666.7 μg/mL

76/384

1.012

89

82

222.2 μg/mL

57/384

1.192

102

88

74.07 μg/mL

68/384

1.082

99

98

24.69 μg/mL

97/384

0.860

88

76

8.23 μg/mL

64/384

1.120

97

94

Vehicle control

64/384

1.120

100

100

Vehicle control for CP

98/384

0.854

65

82

Untreated control

61/384

1.150

100

89

Positive control (CP)

270/384

0.220

18

13

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results Assay 1 (IL)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

Relative Survival§

(%RS)

Relative Total Growth

(%RTG)

-

3

A1

2000 μg/mL

75/384

1.021

100

67

666.7 μg/mL

82/384

0.965

103

79

222.2 μg/mL

73/384

1.038

103

76

74.07 μg/mL

91/384

0.900

95

77

24.69 μg/mL

70/384

1.064

97

83

8.23 μg/mL

83/384

0.957

92

73

Vehicle control

69/384

1.073

100

100

Vehicle control for NQO

83/384

0.957

101

83

Untreated control

75/384

1.021

102

95

Positive control (NQO)

170/384

0.509

45

26

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results Assay 2 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

Relative Survival§

(%RS)

Relative Total Growth

(%RTG)

+

3

A2

2000 μg/mL

53/384

1.238

134

93

666.7 μg/mL

45/384

1.340

158

82

222.2 μg/mL

61/384

1.150

129

76

74.07 μg/mL

86/384

0.935

115

88

24.69 μg/mL

60/384

1.160

125

84

8.23 μg/mL

92/384

0.893

88

67

Vehicle control

86/384

0.935

100

100

Vehicle control for CP

77/384

1.004

103

89

Untreated control

101/384

0.835

84

84

Positive control (CP)

229/384

0.323

29

12

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results Assay 2 (IL)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

Relative Survival§

(%RS)

Relative Total Growth

(%RTG)

-

24

A2

2000 μg/mL

81/384

0.973

122

123

666.7 μg/mL

82/384

0.965

120

124

222.2 μg/mL

98/384

0.854

103

86

74.07 μg/mL

91/384

0.900

98

88

24.69 μg/mL

88/384

0.921

110

111

8.23 μg/mL

82/384

0.965

109

114

Vehicle control

84/384

0.950

100

100

Vehicle control for NQO

107/384

0.799

76

88

Untreated control

68/384

1.082

108

124

Positive control (NQO)

141/384

0.626

36

20

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

 

Summary Tables of Viability Data

 

Viability Results Assay 1 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

+

3

A1

2000 μg/mL

62/384

1.140

666.7 μg/mL

91/384

0.900

222.2 μg/mL

67/384

1.091

74.07 μg/mL

86/384

0.935

24.69 μg/mL

73/384

1.038

8.23 μg/mL

87/384

0.928

Vehicle control

63/384

1.130

Vehicle control for CP

72/384

1.046

Untreated control

83/384

0.957

Positive control (CP)

186/384

0.453

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

 

Viability Results Assay 1 (IL)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

-

3

A1

2000 μg/mL

122/384

0.717

666.7 μg/mL

99/384

0.847

222.2 μg/mL

105/384

0.810

74.07 μg/mL

92/384

0.893

24.69 μg/mL

79/384

0.988

8.23 μg/mL

93/384

0.886

Vehicle control

87/384

0.928

Vehicle control for NQO

83/384

0.957

Untreated control

86/384

0.935

Positive control (NQO)

141/384

0.626

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

 

Viability Results Assay 2 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

+

3

A2

2000 μg/mL

92/384

0.893

666.7 μg/mL

97/384

0.860

222.2 μg/mL

83/384

0.957

74.07 μg/mL

79/384

0.988

24.69 μg/mL

104/384

0.816

8.23 μg/mL

93/384

0.886

Vehicle control

81/384

0.973

Vehicle control for CP

74/384

1.029

Untreated control

79/384

0.988

Positive control (CP)

181/384

0.470

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

 

Viability Results Assay 2 (IL)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Study phase

Test item or control concentration

Number of empty wells/total number of wells

Plating Efficiency

(PE)

-

24

A2

2000 μg/mL

106/384

0.805

666.7 μg/mL

96/384

0.866

222.2 μg/mL

116/384

0.748

74.07 μg/mL

107/384

0.799

24.69 μg/mL

84/384

0.950

8.23 μg/mL

84/384

0.950

Vehicle control

107/384

0.799

Vehicle control for NQO

86/384

0.935

Untreated control

82/384

0.965

Positive control (NQO)

136/384

0.649

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)

 

Summary Tables of Mutagenicity Data

 

Mutagenicity Results of Assay 1 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

+

3

2000 μg/mL

628/768

60/768

80/768

0.712

88.3

666.7 μg/mL

649/768

67/768

52/768

1.102

93.5

222.2 μg/mL

637/768

51/768

80/768

0.510

85.7

74.07 μg/mL

655/768

60/768

53/768

0.446

85.1

24.69 μg/mL

661/768

59/768

48/768

0.001

72.2

8.23 μg/mL

664/768

52/768

52/768

0.116

78.4

Vehicle control

653/768

64/768

51/768

--

71.8

Vehicle control for CP

655/768

51/768

62/768

--

76.1

Untreated control

642/768

66/768

60/768

--

93.6

Positive control

(CP: 4 μg/mL)

156/768

221/768

391/768

●●

1.08E-15

1759.1*

In linear trend analysisβ2/var (β) = 0.68, not significant

* = Statistically significant

● = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/car(Dn) >5.24(at p<0.05).

●● = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of does “n” and that of the vehicle control

var(Dn) = variance of Dn                    β = slope of the curve                        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

Note: Mutation frequency refers to 106viable cells

 

Mutagenicity Results of Assay 1 (IL)

(3-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

-

3

2000 μg/mL

669/768

57/768

42/768

0.104

96.3

666.7 μg/mL

664/768

61/768

43/768

0.010

85.9

222.2 μg/mL

655/768

56/768

57/768

0.165

98.2

74.07 μg/mL

678/768

52/768

38/768

0.724

69.8

24.69 μg/mL

654/768

52/768

62/768

0.097

81.3

8.23 μg/mL

687/768

44/768

37/768

1.440

62.9

Vehicle control

652/768

58/768

58/768

--

88.2

Vehicle control for NQO

675/768

51/768

42/768

--

67.4

Untreated control

659/768

53/768

56/768

--

81.8

Positive control

(NQO: 015 μg/mL)

218/768

254/768

296/768

●●

5.50E-13

1005.5*

In linear trend analysisβ2/var (β) = 1.18, not significant

* = Statistically significant

● = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/car(Dn) >5.24(at p<0.05).

●● = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of does “n” and that of the vehicle control

var(Dn) = variance of Dn                    β = slope of the curve                        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

Note: Mutation frequency refers to 106viable cells

 

Mutagenicity Results of Assay 2 (L)

(3-hour treatment in the presence of S9-mix)

S9-mix

Treatment period

(hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

+

3

2000 μg/mL

655/768

59/768

54/768

0.006

89.1

666.7 μg/mL

644/768

70/768

54/768

0.402

102.4

222.2 μg/mL

624/768

83/768

61/768

0.786

108.4

74.07 μg/mL

671/768

47/768

50/768

0.873

68.3

24.69 μg/mL

659/768

61/768

48/768

0.076

93.7

8.23 μg/mL

657/768

52/768

59/768

0.001

88.1

Vehicle control

648/768

58/768

62/768

--

87.3

Vehicle control for CP

649/768

62/768

57/768

--

81.8

Untreated control

620/768

76/768

72/768

--

108.3

Positive control

(CP: 4 μg/mL)

247/768

165/768

356/768

●●

3.99E-08

1206.6*

In linear trend analysisβ2/var (β) = 0.12, not significant

* = Statistically significant

● = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/car(Dn) >5.24(at p<0.05).

●● = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of does “n” and that of the vehicle control

var(Dn) = variance of Dn                    β = slope of the curve                        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

Note: Mutation frequency refers to 106viable cells

 

Mutagenicity Results of Assay 2 (IL)

(24-hour treatment in the absence of S9-mix)

S9-mix

Treatment period

(hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/total number of wells

Dn2/var(Dn)

Mutation frequency

-

24

2000 μg/mL

665/768

43/768

60/768

0.119

89.5

666.7 μg/mL

661/768

49/768

58/768

0.228

86.6

222.2 μg/mL

622/768

68/768

78/768

2.311

140.9

74.07 μg/mL

634/768

69/768

65/768

0.712

120.0

24.69 μg/mL

659/768

62/768

47/768

0.585

80.6

8.23 μg/mL

681/768

51/768

36/768

2.700

63.3

Vehicle control

657/768

63/768

48/768

--

97.7

Vehicle control for NQO

648/768

56/768

64/768

--

90.8

Untreated control

636/768

78/768

54/768

--

97.7

Positive control

(NQO: 0.1 μg/mL)

267/768

265/768

236/768

●●

8.29E-11

814.3*

In linear trend analysisβ2/var (β) = 0.06, not significant

* = Statistically significant

● = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/car(Dn) >5.24(at p<0.05).

●● = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn= Difference of log mutant frequency of does “n” and that of the vehicle control

var(Dn) = variance of Dn                    β = slope of the curve                        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = 1% methyl cellulose

Negative (vehicle) control for NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

Note: Mutation frequency refers to 106viable cells

 

Historical Control Data

(updated 11 January 2017)

Mutation Frequency of the Negative Controls (2006-2016)

 

Culture medium

Distilled water

Treatments

3h,S9+

3h,S9-

24h,S9-

3h,S9+

3h,S9-

24h,S9-

Average

SD

Min.

Max.

n

94.3

26.9

39.3

198.5

84

103.6

35.3

52.6

235.6

43

106.4

27.4

41.7

179.1

44

90.4

22.7

33.4

121.8

26

96.6

19.0

55.1

125.0

13

96.3

24.6

43.2

141.1

13

 

Dimethyl sulfoxide (DMSO)

 

Treatment

3h,S9+

3h,S9-

24h,S9-

 

 

 

Average

SD
Min.

Max.

n

97.3

33.7

44.2

269.9

101

97.3

38.5

33.7

261.6

57

98.9

26.8

47.1

159.4

50

 

 

 

Mutation Frequency of the Positive Controls (2006-2016)

 

Cyclophosphamide

4-Nitroquinoline-N-oxide

Treatment

3h,S9+

 

 

 

3h,S9-

24h,S9-

Average

SD

Min.

Max.

n

1178.7

524.7

196.1

2642.5

106

 

 

 

722.2

330.0

223.5

1687.3

58

831.9

337.2

245.0

1577.6

52

h = hour

SD = Standard Deviation

S9+ = experiment with metabolic activation

S9- = experiment without metabolic activation

n = number of cases

Conclusions:
The Mouse Lymphoma Assay with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.
In this Mouse Lymphoma Assay, treatment with the test item did not result in a statistically significant and biologically relevant increase in the mutation frequency both in the presence and absence of a rat metabolic activation system (S9 fraction).
Therefore, no mutagenic activity of the test item was concluded in the performed experiments.

In conclusion, no mutagenic effect of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (+S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

1% (w/v) aqueous methyl cellulose solution was used as vehicle of the test item in this study. The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,

Assay 1, 3-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL.

 

In Assays 1-2, there were no large changes in pH or osmolality after treatment.

Precipitate / minimal amount of precipitate was detected at the evaluated test item concentrations in the final treatment medium at the end of the treatment.

 

In Assay 1, following a 3-hour treatment with metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 97%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this experiment was considered as being negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 67%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was also considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 93%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this result confirmed the negative result observed in the first main test with metabolic activation.

 

In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 123%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result of the first main test without metabolic activation.

 

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, no mutagenic effect of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2017 to 04 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Commission Regulation No 440/2008 of 30 May 2008B.10. "Mutagenicity – In Vitro Mammalian Chromosome Aberration Test" as amended by Commission Regulation 2017/735 of 14 February 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
structural chromosome aberrations in somatic and/or germ cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 31 July 2015
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 °C (for short-term storage) or in liquid nitrogen (long-term storage).
The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Chromosome Aberration Assay 1
The examined concentrations of the test item were 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with and without metabolic activation).
Chromosome Aberration Assay 2
The examined concentrations of the test item were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL (experiment without metabolic activation); and 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with metabolic activation).
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
Vehicle / solvent:
Based on the available solubility information (trial formulations of the test item performed at the Test Facility), 1% (w/v) aqueous methyl cellulose solution (abbreviations of 1% methyl cellulose or 1% MC) at 200 mg/mL concentration was selected for vehicle (solvent) of the study. The vehicle was compatible with the survival of the cells and the S9 activity.

Data of the chemicals used for preparation of the vehicle are shown below:
Name: Methyl cellulose
Supplier: HungaroPharma
Lot No.: 5115851
Appearance: White to off white powder
Expiry date: 27 November 2018
Storage conditions: Room temperature

Name: Distilled water
Supplier: B. Braun Pharmaceuticals SA / HungaroPharma /
Lot No.: 63352Y25-2 / 808 0617
Expiry date: 31 July 2019 / 06 December 2017
Storage conditions: Room temperature
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% (w/v) methyl cellulose aqueous solution (abbreviated as 1% MC or 1% Methyl cellulose)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
EXTERNAL METABOLIC ACTIVATION SYSTEM
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
Many substances only develop mutagenic potential after they are metabolised.
Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

Induction of Rat Liver Enzymes
Male Wistar rats (389-479 g animals were 14-17 weeks old at the initiation of E12590) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation dates of the induction of liver enzymes used for preparation S9 used in this study was 16 January 2017 (Citoxlab code: E12590).

Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 19 January 2017 (Citoxlab code: E12590).
The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used in the study was determined to be 29.6 g/L (Citoxlab code: E12590). The sterility of the preparation was confirmed in each case.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

TEST PROCEDURE
Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, two assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments.
Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix); cells were harvested 20-hour after the beginning of the treatment.
In Assay 2, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.

Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5E+5 cells/dish concentration and incubated for approximately 24 hours at 37 °C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 Ml treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, negative (vehicle) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37 °C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL).
The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.

Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
The metaphase analysis was conducted under the control of the Principal Investigator.
When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
At least 150 metaphases with 22 ± 2 chromosomes (centromeres) from each culture (replicate) will be examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) will be recorded separately.

The aberrations are defined in the following way:
Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.
Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc).
Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.
The vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation.
Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.
Statistics:
For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
VEHICLE AND CONCENTRATION SELECTION
Based on the available solubility information (trial formulations of the test item performed at the Test Facility), 1% (w/v) aqueous methyl cellulose solution (abbreviations of 1% methyl cellulose or 1% MC) at 200 mg/mL concentrations was selected for vehicle (solvent) of the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions (up to the maximum recommended concentration and/or up to the solubility limit).

CHROMOSOME ABERRATION ASSAYS
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with and without metabolic activation).
In Assay 1, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 666.7-74.07 μg/mL concentration range and in the 24.69-2.74 μg/mL concentration range (minimal amount of precipitate) with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 74.07, 24.69, and 8.23 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL (experiment without metabolic activation); and 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with metabolic activation).
In Assay 2, similarly to the first experiment, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 666.7-74.07 μg/mL concentration range with metabolic activation and in the 2000-222.2 μg/mL concentration range without metabolic activation. And insolubility (minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 24.69-2.74 μg/mL concentration range with metabolic activation and in the 74.07-8.23 μg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in nay samples of this assay. Therefore, concentrations of 74.07, 24.69, and 8.23 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 222.2, 74.07, and 24.69 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases (1-3) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate].
One endoreduplicated metaphases were detected in the performed experiments.

VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and without metabolic activation; while cytotoxicity was not detected in any experiment with and without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered the range from insolubility to little insolubility.
Three test item concentrations were evaluated in each experiment.
Historical control data are presented in Appendix 6. The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were in harmony the general historical control range of the testing laboratory.
In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations demonstrating the sensitivity of the test system in each assay.
The study was considered to be valid.

Summarized results of the Concentration Selection Cytotoxicity Assay A experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

-

3/20

8.83E+06

111

normal / normal (pH: 7.2; osm: 342 mmol/kg)

Negative (vehicle control)

-

-

3/20

8.03E+06

100

normal / normal (pH: 7.2; osm: 339 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

2000

-

3/20

8.13E+05

101

precipitate / precipitate (pH: 7.2; osm: 341 mmol/kg)

1000

-

3/20

6.98E+05

86

precipitate / precipitate (pH: 7.2; osm: 343 mmol/kg)

500

-

3/20

7.88E+06

98

precipitate / precipitate (pH: 7.2; osm: 341 mmol/kg)

250

-

3/20

8.13E+06

101

precipitate / precipitate (pH: 7.2; osm: 341 mmol/kg)

125

-

3/20

7.70E+06

96

precipitate / precipitate (pH: 7.2; osm: 344 mmol/kg)

62.5

-

3/20

8.18E+06

102

precipitate / precipitate (pH: 7.2; osm: 340 mmol/kg)

31.25

-

3/20

8.40E+06

105

precipitate#/ precipitate#(pH: 7.2; osm: 340 mmol/kg)

15.625

-

3/20

8.48E+06

106

precipitate#/ precipitate#(pH: 7.2; osm: 342 mmol/kg)

7.813

-

3/20

8.68E+06

109

precipitate#/ precipitate#(pH: 7.2; osm: 342 mmol/kg)

3.906

-

3/20

8.73E+06

109

precipitate#/ precipitate#(pH: 7.2; osm: 345 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                          osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay A experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

+

3/20

7.73E+06

96

normal / normal (pH: 7.2; osm: 343 mmol/kg)

Negative (vehicle control)

-

+

3/20

8.05E+06

100

normal / normal (pH: 7.2; osm: 336 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

2000

+

3/20

8.10E+05

101

precipitate / precipitate (pH: 7.2; osm: 340 mmol/kg)

1000

+

3/20

6.80E+05

83

precipitate / precipitate (pH: 7.2; osm: 336 mmol/kg)

500

+

3/20

7.08E+06

87

precipitate / precipitate (pH: 7.2; osm: 337 mmol/kg)

250

+

3/20

7.10E+06

87

precipitate / precipitate (pH: 7.2; osm: 340 mmol/kg)

125

+

3/20

7.00E+06

86

precipitate / precipitate (pH: 7.2; osm: 341 mmol/kg)

62.5

+

3/20

7.25E+06

89

precipitate / precipitate (pH: 7.2; osm: 341 mmol/kg)

31.25

+

3/20

7.30E+06

90

precipitate#/ precipitate#(pH: 7.2; osm: 341 mmol/kg)

15.625

+

3/20

7.68E+06

95

precipitate#/ precipitate#(pH: 7.2; osm: 339 mmol/kg)

7.813

+

3/20

8.00E+06

99

precipitate#/ precipitate#(pH: 7.2; osm: 340 mmol/kg)

3.906

+

3/20

7.80E+06

97

precipitate#/ precipitate#(pH: 7.2; osm: 343 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay B experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

-

20/20

8.28E+06

106

normal / normal (pH: 7.0; osm: 345 mmol/kg)

Negative (vehicle control)

-

-

20/20

7.85E+06

100

normal / normal (pH: 7.0; osm: 344 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

2000

-

20/20

7.35E+05

93

precipitate / precipitate (pH: 7.0; osm: 344 mmol/kg)

1000

-

20/20

7.73E+05

98

precipitate / precipitate (pH: 7.0; osm: 343 mmol/kg)

500

-

20/20

7.40E+06

94

precipitate#/ precipitate#(pH: 7.0; osm: 342 mmol/kg)

250

-

20/20

7.60E+06

97

precipitate#/ precipitate#(pH: 7.0; osm: 345 mmol/kg)

125

-

20/20

6.73E+06

85

precipitate#/ precipitate#(pH: 7.0; osm: 344 mmol/kg)

62.5

-

20/20

6.43E+06

81

precipitate#/ precipitate#(pH: 7.0; osm: 343 mmol/kg)

31.25

-

20/20

7.50E+06

95

precipitate#/ precipitate#(pH: 7.0; osm: 346 mmol/kg)

15.625

-

20/20

8.03E+06

102

precipitate#/ precipitate#(pH: 7.0; osm: 346 mmol/kg)

7.813

-

20/20

7.50E+06

95

precipitate#/ precipitate#(pH: 7.0; osm: 345 mmol/kg)

3.906

-

20/20

7.85E+06

100

precipitate#/ precipitate#(pH: 7.0; osm: 352 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                      #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                         osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity Results of Chromosome Aberration Assay 1 experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

-

3/20

6.28E+06

81

normal / normal (pH: 7.2; osm: 355 mmol/kg)

Negative (vehicle control)

-

-

3/20

7.15E+06

100

normal / normal (pH: 7.2; osm: 350 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

666.7

-

3/20

6.58E+06

88

precipitate / precipitate (pH: 7.2; osm: 353 mmol/kg)

222.2

-

3/20

6.53E+06

87

precipitate / precipitate (pH: 7.2; osm: 346 mmol/kg)

74.07

-

3/20

6.43E06

84

precipitate / precipitate (pH: 7.2; osm: 351 mmol/kg)

24.69

-

3/20

6.53E+06

87

precipitate#/ precipitate#(pH: 7.2; osm: 367 mmol/kg)

8.23

-

3/20

6.58E+06

88

precipitate#/ precipitate#(pH: 7.2; osm: 347 mmol/kg)

2.74

-

3/20

6.60E+06

88

precipitate#/ precipitate#(pH: 7.2; osm: 349 mmol/kg)

Positive control (1 μL/mL EMS)

-

-

3/20

4.90E+06

52

normal / normal (pH: 7.2; osm: 365 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                           osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity Results of Chromosome Aberration Assay 1 experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

+

3/20

5.93E+06

114

normal / normal (pH: 7.2; osm: 346 mmol/kg)

Negative (vehicle control)

-

+

3/20

5.50E+06

100

normal / normal (pH: 7.2; osm: 347 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

666.7

+

3/20

6.05E+06

118

precipitate / precipitate (pH: 7.2; osm: 346 mmol/kg)

222.2

+

3/20

6.08E+06

119

precipitate / precipitate (pH: 7.2; osm: 340 mmol/kg)

74.07

+

3/20

6.00E+06

117

precipitate / precipitate (pH: 7.2; osm: 345 mmol/kg)

24.69

+

3/20

5.88E+06

112

precipitate#/ precipitate#(pH: 7.2; osm: 350 mmol/kg)

8.23

+

3/20

6.48E+06

132

precipitate#/ precipitate#(pH: 7.2; osm: 345 mmol/kg)

2.74

+

3/20

6.28E+06

126

precipitate#/ precipitate#(pH: 7.2; osm: 343 mmol/kg)

Positive control (6 μg/mL CP)

-

+

3/20

3.70E+06

40

normal / normal (pH: 7.2; osm: 356 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                            osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity Results of Chromosome Aberration Assay 2 experiment without metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

-

20/20

8.05E+06

107

normal / normal (pH: 7.4; osm: 347 mmol/kg)

Negative (vehicle control)

-

-

20/20

7.65E+06

100

normal / normal (pH: 7.4; osm: 345 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

2000

-

20/20

7.55E+06

98

precipitate / precipitate (pH: 7.4; osm: 342 mmol/kg)

666.7

-

20/20

7.55E+06

98

precipitate / precipitate (pH: 7.4; osm: 345 mmol/kg)

222.2

-

20/20

8.00E+06

106

precipitate / precipitate (pH: 7.4; osm: 341 mmol/kg)

74.07

-

20/20

7.20E+06

92

precipitate / precipitate#(pH: 7.4; osm: 342 mmol/kg)

24.69

-

20/20

8.05E+06

107

precipitate#/ precipitate#(pH: 7.4; osm: 344 mmol/kg)

8.23

-

20/20

7.85E+06

104

precipitate#/ precipitate#(pH: 7.4; osm: 344 mmol/kg)

Positive control (0.4 μL/mL EMS)

-

-

20/20

5.25E+06

57

normal / normal (pH: 7.4; osm: 354 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                          osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity Results of Chromosome Aberration Assay 2 experiment with metabolic activation

Test group

Dose (μg/mL)

S9-mix

Treatment / sampling time

Cell number (total)

RICC (%)*

Observations beginning / end of treatment

Untreated control

-

+

3/20

7.95E+06

110

normal / normal (pH: 7.2; osm: 339 mmol/kg)

Negative (vehicle control)

-

+

3/20

7.40E+06

100

normal / normal (pH: 7.2; osm: 336 mmol/kg)

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

666.7

+

3/20

6.50E+06

83

precipitate / precipitate (pH: 7.2; osm: 336 mmol/kg)

222.2

+

3/20

7.55E+06

103

precipitate / precipitate (pH: 7.2; osm: 333 mmol/kg)

74.07

+

3/20

7.30E+06

98

precipitate / precipitate (pH: 7.2; osm: 336 mmol/kg)

24.69

+

3/20

7.35E+06

99

precipitate / precipitate#(pH: 7.2; osm: 338 mmol/kg)

8.23

+

3/20

7.60E+06

104

precipitate#/ precipitate#(pH: 7.2; osm: 333 mmol/kg)

2.74

+

3/20

7.35E+06

99

precipitate#/ precipitate#(pH: 7.2; osm: 336 mmol/kg)

Positive control (6 μg/mL CP)

-

+

3/20

4.60E+06

47

normal / normal (pH: 7.2; osm: 339 mmol/kg)

*: compared to the negative (vehicle) control (1% methyl cellulose)                                                       #: minimal amount

RICC: Relative Increase in Cell Counts                                                                                          osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate#

Mean % aberrant cells###

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] without metabolic activation (-S9)

Untreated control

3h / 20h

81

-

NE

Negative (vehicle) control [300]

3h / 20h

100

-

2.3

666.7 μg/mL

3h / 20h

88

+

NE

222.2 μg/mL

3h / 20h

87

+

NE

74.07 μg/mL [300]

3h / 20h

84

+

2.7

24.69 μg/mL [300]

3h / 20h

87

+a

3.0

8.23 μg/mL [300]

3h / 20h

88

+a

3.3

2.74 μg/mL

3h / 20h

88

+a

NE

Positive control [300]

3h / 20h

52

-

12.3**

Negative (vehicle) control: (1% methyl cellulose)

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of precipitate

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate#

Mean % aberrant cells###

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] without metabolic activation (-S9)

Untreated control

3h / 20h

114

-

NE

Negative (vehicle) control [300]

3h / 20h

100

-

3.0

666.7 μg/mL

3h / 20h

118

+

NE

222.2 μg/mL

3h / 20h

119

+

NE

74.07 μg/mL [300]

3h / 20h

117

+

2.7

24.69 μg/mL [300]

3h / 20h

112

+a

2.7

8.23 μg/mL [300]

3h / 20h

132

+a

4.3

2.74 μg/mL

3h / 20h

126

+a

NE

Positive control [54]

3h / 20h

40

-

92.6***

Negative (vehicle) control: (1% methyl cellulose)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of precipitate

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate#

Mean % aberrant cells###

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] without metabolic activation (-S9)

Untreated control

20h / 20h

107

-

NE

Negative (vehicle) control [300]

20h / 20h

100

-

4.0

2000 μg/mL

20h / 20h

98

+

NE

666.7 μg/mL

20h / 20h

98

+

NE

222.2 μg/mL

20h / 20h

106

+

4.3

74.07 μg/mL [300]

20h / 20h

92

+a

1.7

24.69 μg/mL [300]

20h / 20h

107

+a

3.3

8.23 μg/mL [300]

20h / 20h

104

+a

NE

Positive control [118]

20H / 20H

57

-

42.4***

Negative (vehicle) control: (1% methyl cellulose)

Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of precipitate

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 2 with metabolic activation

Concentration (μg/mL)

[Number of analyzed cells]

Time of Treatment / Sampling

RICC#(%)

Precipitate#

Mean % aberrant cells###

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] without metabolic activation (-S9)

Untreated control

3h / 20h

110

-

NE

Negative (vehicle) control [300]

3h / 20h

100

-

4.7

666.7 μg/mL

3h / 20h

83

+

NE

222.2 μg/mL

3h / 20h

103

+

NE

74.07 μg/mL [300]

3h / 20h

98

+

3.7

24.69 μg/mL [300]

3h / 20h

99

+a

7.0

8.23 μg/mL [300]

3h / 20h

104

+a

4.0

2.74 μg/mL

3h / 20h

99

+a

NE

Positive control [62]

3h / 20h

47

-

80.6***

Negative (vehicle) control: (1% methyl cellulose)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: minimal amount of precipitate

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

HISTORICAL CONTROL DATA

3h/20h treatment/sampling time without S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(EMS)

Incl. Gaps

Excel. Gaps

Incl. Gaps

Excl. Gaps

Mean

2.71

1.20

22.67

18.02

SD

1.65

0.82

12.43

8.16

Range

0-7

0-3

4-63

4-40

n

46

46

40

40

 

3h/20h treatment/sampling time with S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(CP)

Incl. Gaps

Excel. Gaps

Incl. Gaps

Excl. Gaps

Mean

3.24

1.45

73.00

69.50

SD

1.57

0.95

23.51

25.64

Range

0-8

0-4

21-100

21-100

n

43

43

21

21

 

SD = standard deviation

Range = min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

 

Notes:

1. In the period of 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.

2. In studies performed before the updated OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for a positive control sample was halted when 15 aberrant cells were counted.

 

20h/28h treatment/sampling time without S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(EMS)

Incl. Gaps

Excel. Gaps

Incl. Gaps

Excl. Gaps

Mean

2.58

1.13

34.97

30.96

SD

1.81

0.86

10.94

9.80

Range

0-8

0-4

5-76

5-68.2

n

43

43

43

43

 

3h/28h treatment/sampling time with S9-mix

 

Aberration rate

(phases with aberration in %)

Negative control

Positive control

(CP)

Incl. Gaps

Excel. Gaps

Incl. Gaps

Excl. Gaps

Mean

3.07

1.47

52.63

48.80

SD

1.64

1.07

23.98

24.49

Range

0-8

0-5

7-93.8

6-93.8

n

43

43

21

21

 

SD = standard deviation

Range = min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

 

Notes:

1. In the period of 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.34 (including gaps) and 18.36 (excluding gaps) in 22 experiments.

2. In studies performed before the updated OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for a positive control sample was halted when 15 aberrant cells were counted.

3. These historical controls were used for this study due to the lack of the 20h/20h treatment/sampling time without S9-mix historical control.

Conclusions:
The test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant, reproducible, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was considered as not clastogenic in this test system.
Executive summary:

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in 1% MC and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.

 

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with and without metabolic activation).

 

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 666.7-74.07 μg/mL concentration range (precipitate) and in the 24.69-2.74 μg/mL concentration range (minimal amount of precipitate) with and without metabolic activation. There were no large changes in the pH and osmolality.

No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 74.07, 24.69, and 8.23 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

 

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL (experiment without metabolic activation); and 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL (experiment with metabolic activation).

 

In Assay 2, similarly to the first experiment, insolubility (precipitate) was detected at the end of the treatment period in the final treatment medium in the 666.7-74.07 μg/mL concentration range with metabolic activation and in the 2000-222.2 μg/mL concentration range without metabolic activation. And insolubility (minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 24.69-2.74 μg/mL concentration range with metabolic activation and in the 74.07-8.23 μg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in nay samples of this assay. Therefore, concentrations of 74.07, 24.69, and 8.23 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations 222.2, 74.07, and 24.69 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.

 

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

 

Polyploid metaphases (1-3) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate].

One endoreduplicated metaphases were detected in the performed experiments.

 

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

In conclusion, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was considered as not clastogenic in this test system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro: Ames Assay

The aim of the study was to assess the mutagenic potential of the test material Propanoic acid, 3-(dodecylthio)-,2,2-bis[[3-dodecylthio)-1-oxopropoxy]methyl]-1,3-propanediyl ester using a bacterial/microsome test system. The study was based on the in vitro technique Ames test which mutagenic activity was assess by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of E-coli to various concentrations of the test material. This was done in accordance to the following guidelines:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

EU Method B13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria).

The test material was considered to be non-mutagenic under the conditions of the study.

Genetic toxicity in vitro: Mouse lymphoma assay

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (+S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

In Assay 1, following a 3-hour treatment with metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 97%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this experiment was considered as being negative.

In Assay 1, following a 3-hour treatment without metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 67%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was also considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 93%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this result confirmed the negative result observed in the first main test with metabolic activation.

In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 2000 μg/mL (relative total growth of 123%) and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result of the first main test without metabolic activation.

 

In conclusion, no mutagenic effect of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Genetic toxicity in vitro: Chromosome aberration

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells.

 

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

Polyploid metaphases (1-3) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate].

One endoreduplicated metaphases were detected in the performed experiments.

 

In conclusion, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was considered as not clastogenic in this test system.

Justification for classification or non-classification