Dioctadecyl 3,3'-thiodipropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, GLP: negative

OECD 473, GLP (V79 cells): negative

OECD 490, GLP: negative

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

thymidine kinase (TK) locus

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)

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

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

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.

Conclusions:

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-09-29 - 1998-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1986

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from liver of phenobarbital and b-naphthoflavone induced rats

Test concentrations with justification for top dose:

3, 6, 30, and 300 µg/mL (without activation); 3, 5, 10, 20, and 40 µg/mL (with activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
The test item was added to the culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. After 3 x 10 minutes of ultrasonic treatment, a homogenous suspension could be prepared. Using higher concentrations, small test article particles were observed, leading to an inhomogenous suspension.

DURATION
- Preincubation period: at least 48 h
- Exposure duration: First experiment: 4h with and without S9 mix; Second experiment: 4h with metabolic activation and 18h and 28 h without metabolic activation
- Expression time (cells in growth medium): Both 18 and 28 h for the first and second experiment, each with and without S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Cell numbers of two cultures (10 coordinate defined fields per culture) were determined for each experimental group.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance were considered together

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 6 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. The highest applied concentration in the pre-test was chosen with regard to the properties of the formulation of the test article. Test article concentrations between 1 - 300 µg/mL (without S9 mix) or 1 - 285 µg/mL (with S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers to 55.0 % of control were observed after treatment with 300 µg/mL whereas in the presence of S9 mix no toxic effects were observed. Precipitation of the test article was observed 4 h after start of treatment at concentrations of 5 µg/mL and above in the absence of S9-mix and at 10 µg/mL and above in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In the range

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

SUMMARY OF RESULTS

						Aberrant cells in %
Exp	Preparation Interval	concentration (µg/ml)	S9 Mix	Polyploid cells (%)	Mitotic indices in % of control	incl. Gaps	excl. Gaps*	with exchanges
I	18 h	solvent control	-	2.5	100.0	3.5	2.0	1.0
	18 h	1.5	-	4.0	108.1	2.5	2.0	0.0
	18 h	3.0	-	4.0	105.2	1.5	0.5	0.0
	18 h	6.0P	-	4.0	98.4	2.5	1.0	0.0
	18 h	300.0P	-	2.0	120.2	4.0	3.0	0.5
II	18 h	solvent control	-	3.0	100.0	3.5	3.5	0.5
	18 h	1.5	-	2.0	116.1	3.5	2.5	0.0
	18 h	3.0	-	1.5	84.4	3.0	2.0	0.5
	18 h	6.0P	-	1.5	94.7	2.5	1.5	0.0
	18 h	30.0P	-	2.5	95.6	0.0	0.0	0.0
I	28 h	solvent control	-	3.0	100.0	1.5	1.0	0.0
	28 h	3.0	-	2.0	128.5	3.5	1.5	0.0
	28 h	30.0P	-	3.5	111.5	2.0	0.5	0.0
II	28 h	solvent control	-	2.0	100.0	1.5	1.0	0.0
	28 h	6.0P	-	3.0	83.3	2.0	1.0	0.0
	28 h	30.0P	-	4.5	87.4	2.5	2.5	0.0
I	18 h	solvent control	+	3.0	100.0	4.0	2.0	0.0
	18 h	3.0	+	1.5	85.9	2.0	2.0	0.0
	18 h	5.0	+	2.0	67.7	3.5	1.5	0.0
	18 h	10.0	+	3.0	71.8	5.0	4.0	0.5
	18 h	20.0P	+	4.0	73.5	2.5	1.5	0.0
II	18 h	solvent control	+	2.0	100.0	1.5	1.5	1.0
	18 h	3.0	+	2.5	93.6	4.0	3.0	0.0
	18 h	5.0	+	2.0	88.3	3.5	3.0	0.5
	18 h	10.0P	+	4.0	109.0	2.5	1.5	0.5
	18 h	20.0P	+	2.0	105.8	3.0	1.5	1.0
I	28 h	solvent control	+	2.0	100.0	1.5	1.0	0.0
	28 h	10.0	+	2.5	143.8	1.5	0.0	0.0
	28 h	20.0P	+	4.5	123.3	0.0	0.0	0.0
II	28 h	solvent control	+	3.5	100.0	2.0	2.0	1.0
	28 h	10.0P	+	4.5	85.6	1.0	0.5	0.0
	28 h	40.0P	+	3.5	89.7	1.5	1.5	0.0

* inclusive cells carrying exchanges

P Precipitation occurred

Aberrant cells in the positive control groups: 11.0 % - 31.0 %

Conclusions:

The test article is considered to be non-mutagenic in this chromosome aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18-Nov-2021 to 16-Dec-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Preparation of S9-Mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter-sterilized (0.22 μm). To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

First Mutation Experiment:
TA100 & WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 2500 μg/plate
Justication: test item solubility (the test item was only soluble up to 50 mg/mL which resulted in the highest concentration of 2500 μg/plate)
TA1535: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
Justication: formation of precipitation (Based on the results of the dose-range finding test at which precipitate was observed at and above the concentration of 17 μg/plate)
TA1537 & TA98: 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate
Justication: formation of precipitation (no precipitate or cytotoxicity was observed at the highest concentration tested. Therefore, an additional experiment was performed in these tester strains at concentrations of 512 and 1600 μg/plate)

Second Mutation Experiment:
1, 10, 50, 100, 500 and 1500 μg/plate
Justification: Based on First Mutation Experiment

Vehicle / solvent:

Tetrahydrofuraan (THF)

Justification: The test item proved insoluble in Milli-Q water, DMSO and ethanol. The test item formed a clear colorless solution in THF up to 50 mg/mL, resulting in a final top concentration of 2500 μg/plate (highest achievable concentration). The stock solution was treated with ultrasonic waves until the test item had completely dissolved.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other:

Remarks:

with metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Remarks:

without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL)
- Test substance added in agar (plate incorporation)


Experimental Design:

Dose-range Finding Test / First Experiment:
Selection of an adequate range of concentrations was based on a dose-range finding test with
the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight
concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 2500 μg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was the
level at which the test item exhibited limited solubility.

Mutation Assay/ Second Experiment:
At least five different concentrations (increasing with approximately half-log steps) of the test
item were tested in triplicate in each strain. The above mentioned dose-range finding study
with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation
experiment. In the second part of this experiment, the test item was tested both in the absence
and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a
follow-up experiment using modified conditions (different S9-mix concentrations), the test
item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
Initially, in the second experiment tester strain TA100 in the presence of 10% S9-mix was
rejected since some of the acceptability criteria were not met. This part of the study was
repeated.
The negative control (vehicle) and relevant positive controls were concurrently tested in each
strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(10^ 9 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in THF or
control solution and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M
phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex
and the content of the top agar tube was poured onto a selective agar plate. After
solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ±
1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for
Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli)
were counted.


The revertant colonies were counted automatically with the Sorcerer Colony Counter.

Evaluation criteria:

A reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared
against relevant historical control data generated at the testing facility.
b) The selected concentration-range should include a clearly toxic concentration or should
exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or
should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen
event. If the results are considered invalid due to contamination, the experiment will be
repeated.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants was
evaluated for its biological relevance including a comparison of the results with the historical
control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in strain TA100 or WP2uvrA is not greater than
2 times the concurrent vehicle control, and the total number of revertants in
strains TA1535, TA1537 or TA98 is not greater than 3 times the concurrent vehicle
control.
b) The negative response should be reproducible in at least 1 follow-up experiment. A result is considered positive in the test if:
a) The total number of revertants in strain TA100 or WP2uvrA is greater than 2
times the concurrent vehicle control, or the total number of revertants in strains
TA1535, TA1537, TA98 is greater than 3 times the concurrent vehicle control.
b) In case a follow up experiment is performed, the positive response should bereproducible
in at least 1 follow up experiment.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test/First Mutation Experiment:

Precipitate:
The test item was tested up to or beyond a precipitating concentration. The test item precipitated heavily on the plates at 2500 μg/plate under both conditions in TA100 and
WP2uvrA and at 1600 μg/plate in the presence of S9-mix in WP2uvrA and TA1535, and in the presence and absence of S9-mix in TA1537 and TA98. Therefore the number of revertants for these concentrations could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested.

Toxicity:
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated in Appendix 2.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity:
In tester strain TA1537 in the presence of S9-mix (at 52 and 164 μg/plate) the test item induced a 3.0-fold increase in the number of revertant colonies compared to the solvent
control. However, this increase was within the historical control data range and related to a relative low solvent control. No increases in the number of revertants were observed at any other tester strains at any of the concentrations tested and therefore this increase is considered not biologically relevant.


Second Mutation Experiment:
Precipitate:
The test item precipitated at concentrations of 1 μg/plate and upwards in tester strain WP2uvrA in the absence of S9-mix, at concentrations of 50 μg/plate and upwards at tester
strains TA98, TA1535 and TA1537 in the absence of S9-mix and in tester strains TA1537, TA100 and WP2uvrA in the presence of S9-mix. Furthermore, the test item precipitated at concentrations of 100 μg/plate and upwards in tester strain TA100 in the absence of S9-mix and in tester strains TA98 and TA1535 in the presence of S9-mix At the concentrations of 500 and/or 1500 μg/plate the test item precipitated heavily on the plates, therefore the number of revertants for these concentrations could not be determined.

Toxicity:
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity:
In the second mutation assay, no increase in the number of revertants was observed upon treatment with Dioctadecyl 3,3’-thiodipropionate under all conditions tested.

Conclusions:

All bacterial strains showed negative responses over the entire concentration-range, i.e. no biologically relevant, concentration-related increase in the number of revertants. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the bacterial reverse mutation assay using strains of Salmonella typhimurium and Escherichia coli under the conditions tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Mutatgenicity in Bacteria

A mutagenicity study in bacteria was performed according to OECD guideline 471 and under GLP. 

In a first study, the substance was tested at a concentration range of 0.55 to 1600 µg/plate, in the absence and presence of metabolic activation in the tester strains TA1535, TA1537 and TA98 as well as TA 100 and E. coli WP2 uvrA. Expect for tester strain TA1535 in the absence of S9-mix which was tested up to 164 μg/plate. Precipitation was observed starting at 17 µg/plate (TA1535), 52 µg/plate (TA100, WP2 uvrA) and 512 µg/plate (TA1537, TA98). At 1600 μg/plate the test item precipitated heavily on the plates, therefore the number of revertants could not be determined for this concentration. Cytotoxicity was not observed at any of the concentrations tested. In a follow-up experiment with additional parameters, the test item was tested at a concentration range of 1 to 1500 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating concentration level. Precipitation was observed starting at the concentration of 1 μg/plate. At the concentration of 500 and/or 1500 μg/plate the test item precipitated heavily on the plates, therefore the number of revertants for these concentrations could not be determined. No cytotoxicity was observed at any of the concentrations tested.
The test item did not induce a biologically relevant, concentration-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a
follow-up experiment using modified experimental conditions.

The findings of this study are consistent with the results of a screening test with Salmonella strains TA 98, TA 100 and TA 1537 that was performed with up to 5 mg/plate of the actual substance (Ciba-Geigy, 1989). The plate incorporation method was applied both with and without the addition of liver postmitochondrial fraction prepared from Arochlor 1254 induced rats and acetone was used as vehicle. With the limitation of the number of tester strains, the procedure and the reporting details are adequate for hazard assessment.

 

Mutagenicity in mammalian cells

The test substance was tested according to OECD guideline 490 and under GLP in L5178Y mouse lymphoma cells for the potential of mutagenicity in mammalian cells. 

The test was performed 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. In the first experiment, the test material was tested up to concentrations of 2.5 μg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. The test material precipitated in the culture medium at this concentration. In the second experiment, the test material was tested up to concentrations of 20 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The test material precipitated in the culture medium at this concentration. 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. The positive control substances methyl methanesulfonate and cyclophosphamide produced significant increases in the mutant frequency which was within the 95% control limits of the distribution of the historical positive control database. 

The test substance did not produce a biologically relevant increase in the mutant frequency either in the presence nor the absence of S9-mix. Therefore, the test substance was considered not mutagenic in the mouse lymphoma assay under the experimental conditions tested. 

 

Chromosomal Damage

The key study for clastogenictiy in mammalian cells in vitro was performed in V79 cells (CCR, 1998). The study was performed according to OECD testing guideline 473 (1983) and under GLP. The test article was formulated in culture medium (MEM). The chromosomes were prepared 18 h and 28 h after start of treatment. The treatment interval was 4 h (exp. I: without and with metabolic activation; exp. II: with metabolic activation) or 18 h and 28 h without metabolic activation (exp. II). In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test was chosen with regard to the properties of the formulation of the test article. A homogenous suspension could be prepared at 300 μg/ml in the absence of S9 mix and 285 μg/ml in the presence of S9 mix. Test article concentrations between 1 and 300 μg/ml (without S9 mix) or 1 and 285 μg/ml (with S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers to 55.0 % of control were observed after treatment with 300 μg/ml whereas in the presence of S9 mix no toxic effects were observed. In the pre-test precipitation of the test article was observed 4 h after start of treatment at concentrations of 5 μg/ml and above in the absence of S9 mix and 10 μg/ml and above in the presence of S9 mix. In experiment I, test article concentrations within a range of 0.75 - 300 μg/ml (without S9 mix) and 0.6-20 μg/ml (with S9 mix) were applied for the investigation of the potential to induce cytogenetic damage. In experiment II, the applied concentration ranges were 0.75 - 300 μg/ml in the absence of S9 mix and 1.0-40 μg/ml in the presence of S9 mix. In the absence and the presence of S9 mix, in both experiments, no reduction of the mitotic index or the cell number was observed, except in the presence of S9 mix in experiment II at interval 28 h after treatment with 40 μg/ml reduced cell numbers were observed. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.