Dioctadecyl 3,3'-thiodipropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Feb-2022 to 24-May-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Dioctadecyl 3,3'-thiodipropionate

EC Number:

211-750-5

EC Name:

Dioctadecyl 3,3'-thiodipropionate

Cas Number:

693-36-7

Molecular formula:

C42H82O4S

IUPAC Name:

dioctadecyl 3,3'-thiodipropionate

Test material form:

solid: flakes

Method

Target gene:

thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells obtained from American Type Culture Collection (ATCC, Manassas, USA) in 2011
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Normal cell cycle time (negative control): n.a.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: -
- Methods for maintenance in cell culture: Cell density was kept below 1x10^6 cells/mL.
- Cell cycle length, doubling time or proliferation index: -
- Modal number of chromosomes: -
- Periodically checked for karyotype stability: -
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic medium was made of RPMI 1640 HEPES buffered medium or RPMI 1640 HEPES buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Growth medium consisted of basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 31.5 - 103.0%, lowest and highest mean values ranging from 84.8% to 99.0%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.1 - 37.6°C, lowest and highest mean values ranging from 34.5°C to 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated.

Cytokinesis block (if used):

-

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2x6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The solution was filter-sterilized (0.22 μm). To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 4% (v/v)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): -

Test concentrations with justification for top dose:

First mutagenicity test:
A dose-range finding test with concentration levels between 16-250 µg/ml was performed. Since the test material was only soluble in THF at 100 mg/mL, this resulted in a maximum feasible concentration of 250 μg/mL in the exposure medium. After 3 and 24 hours, the test material precipitated in the exposure medium at concentrations of 16 μg/mL and above. Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 250 μg/mL compared to the solvent control after 3 hours of treatment with 24 and 48 hours of subculture. No toxicity in the relative suspension growth was observed up to test material concentrations of 125 μg/mL compared to the solvent control after 24 hours of treatment with 24 hours of subculture in the absence of S9 mix.

Mutagenicity Test:
Based on the dose-range finding test, the following concentration levels were used.
First mutagenicity test and 1st repeat test: 0.05. 0.1, 0.2, 0.5, 1, 2, 4, 8, 16 and 32 μg/mL with and without S9-mix

Since no precipitation and no toxicity at any of the concentrations were observed, the following concentration levels were used.
2nd repeat test: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 25, 50 and 100 μg/mL with and without S9-mix

Second mutagenicity test:
Based on the results of the dose-range finding test and experiment 1, the following concentration levels were selected for the second mutagenicity testing.
Second mutagenicity test: 0.15, 0.3, 0.6, 1.3, 2.5, 5, 10, 20, 50 and 100 μg/mL exposure medium without S9 mix

Since the acceptability criteria for the solvent controls were not met, the following concentration levels were used in the repeat experiment.
1st repeat test: 0.15, 0.3, 0.6, 1.3, 2.5, 5, 10, 20, 50 and 100 μg/mL exposure medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a clear colourless solution in THF.

- Justification for percentage of solvent in the final culture medium: 0.25% (v/v)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: test item and positive control were tested in single, solvent was tested in duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): per culture 8 x 10^6 cells (10^6 cells/mL for 3- hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24- hour treatment) were used
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: test item was tested in the presence of S9-mix with a 3- hour treatment period and in the absence of S9-mix with 3- and 24- hour treatment periods
- Harvest time after the end of treatment (sampling/recovery times): cells were cultured for 2 days after the treatment period (during this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): selection using trifluorothymidine (TFT; 5 µg/mL), incubated for 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): none, but staining with MTT for 1.5-2 hours
- Method used: microwell plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the cloning efficacy at day2 (CEday2), the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
- For determination of the mutant frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
The plates for the CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours by adding MTT to aid distinguishing between small and large colonies. The plates were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
The suspension growth (SG) for the 3- hour treatment = [Day 1 cell count/1.6 x 10^5] x [Day 2 cell count/1.25 x 10^5]
The suspension growth (SG) for the 24- hour treatment = [Day 0 cell count/1.25 x 10^5] x [Day 1 cell count/1.25 x 10^5] x [Day 2 cell count/1.25 x 10^5]
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well
The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100
The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CEday2) in the same culture were determined and the mutant frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CEday2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.

Evaluation criteria:

Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent mutation frequency (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 x 10^-6.
A test material is considered positive in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

Acceptability criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutant frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3-hour treatment, and between 32 and 180 for the 24-hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutant frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Additional info on results
TEST-SPECIFIC CONFOUNDING FACTORS
The pH and osmolarity of the culture medium containing the highest tested concentration (if not precipitating) will be recorded as part of the solubility test or as part of this study.
- Data on pH: no effect (pH 7.2 at 16 µg/ml test substance vs. pH 7.2 in control)
- Data on osmolality: no effect (0.327 Osm/kg test substance vs. 0.328 Osm/kg in control)
- Possibility of evaporation from medium: not expected based on vapor pressure of the material
- Water solubility: The test item was dissolved in tetrahydrofuran. The highest concentration (250 µg/mL) in the mutagenicity test was determined based on the solubility of the test material in the culture medium.
- Precipitation and time of the determination: In the dose range finding test, the test material precipitated in the exposure medium at concentrations of 16 μg/mL and above after 3 and 24 hours. In the first mutagenicity test with 3 hours of treatment, precipitation was observed at and above 2.5 μg/mL. In the second mutagenicity test with 24 hours of treatment, precipitation was observed at and above 20 μg/mL.
- Definition of acceptable cells for analysis: -
- Other confounding effects: -

RANGE-FINDING/SCREENING STUDIES (if applicable):
Cells were treated with a test material concentration range of 16 to 250 μg/mL in the absence of S9-mix with 3- and 24- hour treatment periods and in the presence of S9-mix with a 3- hour treatment period. After 3 and 24 hours, the test material precipitated in the exposure medium at concentrations of 16 μg/mL and above. The pH and osmolarity at a concentration of 16 μg/mL were 7.2 and 0.327 Osm/kg respectively (compared to 7.2 and 0.328 Osm/kg in the solvent control). As this concentration (the lowest tested) showed already precipitation but showed no significant difference when compared to the vehicle control, pH and osmolarity were considered sufficiently investigated in the presence of precipitation.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 250 μg/mL compared to the solvent control after 3 hours of treatment and 24 and/or 48 hours of subculture.
No toxicity in the relative suspension growth was observed up to test material concentrations of 125 μg/mL compared to the solvent control after 24 hours of treatment and 24 hours of subculture.
Results are detailed in Table 1 and 2 in the section “Any other information on results incl. tables”

STUDY RESULTS
The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

- Results from cytotoxicity measurements:
In Experiment 1 using 3 hours of treatment with/without S9 mix, there were no signs of toxicity up to the highest concentration tested. Precipitation was observed at and above 2.5 μg/mL.
In Experiment 2 using 24 hours of treatment without S9 mix, there were no signs of toxicity up to the highest concentration tested. Precipitation was observed at and above 20 μg/mL.
- Genotoxicity results:
Both in the First and in the Second Mutagenicity Test, no biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

The results of the First and Second Mutagenicity Test are detailed in Table 3 and 4 in the section “Any other information on results incl. tables”.

HISTORICAL CONTROL DATA: results were compare the available historical control data; please see table 5 and 6 in the section “Any other information on results incl. tables”

Remarks on result:

other: no mutagenic potential with and without metabolic activation

Any other information on results incl. tables

Table 1: Dose-range Finding Test: Cytotoxicity (3 Hour Treatment)

dose (μg/mL)	cell count after 24 hours of subculture (10^5 cells/mL)	cell count after 48 hours of subculture (10^5 cells/mL)	SG	RSG (%)
without metabolic acitvation
SC	3.0	5.2	8	100
16 (1)	4.6	5.4	12	159
31 (1)	4.3	5.6	12	154
63 (1)	3.9	5.5	11	138
125 (1)	3.3	6.1	10	129
250 (1)	4.9	5.8	14	182
with metabolic activation
SC	2.1	4.6	5	100
16 (1)	2.2	6.4	7	146
31 (1)	2.5	5.4	7	140
63 (1)	2.4	5.2	6	129
125 (1)	3.3	5.0	8	171
250 (1)	3.8	5.0	10	197

All calculations were made without rounding off.

SC = solvent control = THF

SG = suspension growth

RSG = relative suspension growth

(1) = the test material precipitated in the exposure medium

SG = Suspension growth = [Cell count after 24 hour of subculture (Day 1) /1.6 x 10^5] x [Cell count after 48 hours of subculture (Day 2) /1.25 x 10^5]

RSG = [SG(test)/SG(control)] x 100

 

Table 2: Dose-range Finding Test: Cytotoxicity (24 Hour Treatment)

dose (μg/mL)	cell count after 24 hours of subculture (10^5 cells/mL)	cell count after 48 hours of subculture (10^5 cells/mL)	SG	RSG (%)
without metabolic acitvation
SC	10.8	4.7	32	100
16 (1)	10.4	3.8	25	78
31 (1)	10.7	4.7	32	99
63 (1)	10.1	4.3	28	86
125 (1)	10.8	4.7	32	100
250 (1)	0.0 (2)	0.0	0	0

All calculations were made without rounding off.

SC = solvent control = THF

SG = suspension growth

RSG = relative suspension growth

(1) = the test material precipitated in the exposure medium

(2) = no subculture performed (less than 1.25 x 10^5 cells/mL)

SG = Suspension growth = [Cell count after 24 hour of subculture (Day 1) /1.6 x 10^5] x [Cell count after 48 hours of subculture (Day 2) /1.25 x 10^5]

RSG = [SG(test)/SG(control)] x 100

 

Table 3: Experiment 1: Cytotoxic and Mutagenic Response in the Mouse Lymphoma L5178Y Test System

dose (μg/mL)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	mutant frequency per 10^6 survivors
					total	( small	large )
without metabolic activation
3 hour treatment
SC	100	97	100	100	72	( 30	39 )
SC		83			68	( 18	49 )
0.08	103	93	103	106	67	( 35	30 )
0.16	103	97	108	111	73	( 22	49 )
0.31	116	98	109	127	72	( 30	40 )
0.63	111	97	108	120	72	( 37	32 )
1.25	101	102	114	115	57	( 17	39 )
2.5 (1)	108	77	86	93	95	( 36	55 )
MMS	77	60	67	52	701	( 204	431 )
with metabolic activation
3 hour treatment
SC	100	86	100	100	64	( 23	39 )
SC		94			68	( 23	44 )
0.08	78	80	89	70	68	( 31	36 )
0.16	91	84	93	84	64	( 37	25 )
0.31	76	95	106	81	50	( 10	40 )
0.63	96	83	92	88	72	( 21	50 )
1.25	94	81	90	85	73	( 30	41 )
2.5 (1)	94	76	84	79	65	( 28	35 )
CP	28	75	83	23	334	( 123	192 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = THF; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = the test item precipitated in the exposure medium

 

Table 4: Experiment 2: Cytotoxic and Mutagenic Response in the Mouse Lymphoma L5178Y Test System

dose (μg/mL)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	mutant frequency per 10^6 survivors
					total	( small	large )
without metabolic activation
24 hour treatment
SC	100	90	100	100	131	( 24	103 )
SC		104			83	( 13	68 )
0.15	91	98	101	93	71	( 22	47 )
0.3	121	99	103	124	102	( 30	67 )
0.6	117	91	94	110	98	( 35	59 )
1.3	103	145	150	155	73	( 15	55 )
2.5	120	110	113	136	88	( 11	75 )
5	103	94	97	99	90	( 20	67 )
10	108	90	93	100	120	( 31	83 )
20 (1)	130	107	110	143	111	( 21	85 )
MMS	107	88	90	96	465	( 79	356 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = THF; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = the test item precipitated in the exposure medium

 

Table 5: Historical Control Data of the Spontaneous Mutant Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutant frequency per 10^6 survivors
	-S9 mix		+ S9 mix
	3-hour treatment	24-hour treatment	3-hour treatment
Mean	101	98	101
SD	28	25	28
n	82	77	82
Lower Control Limit (95% Control Limits)	46	48	47
Upper Control Limit (95% Control Limits)	156	148	156

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between December 2018 and December 2021.

 

Table 6: Historical Control Data of the Mutant Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutant frequency per 10^6 survivors
	-S9 mix		+ S9 mix
	3-hour treatment	24-hour treatment	3-hour treatment
Mean	1021	795	1425
SD	405	235	740
n	79	78	80
Lower Control Limit (95% Control Limits)	227	334	-25
Upper Control Limit (95% Control Limits)	1816	1255	2876

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between December 2018 and December 2021.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test stubstance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions chosen.