Reaction products of 3,3'-thiodi(propionic acid) and alcohols, C11-14-iso-, C13-rich

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 19th to January 27th, 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted May 30th, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted June 26th, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3,3'-thiodi(propionic acid) and alcohols, C11-14-iso-, C13-rich

EC Number:

823-780-1

Cas Number:

1034820-43-3

Molecular formula:

UVCB substance

IUPAC Name:

Reaction products of 3,3'-thiodi(propionic acid) and alcohols, C11-14-iso-, C13-rich

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH (batch: TA98: 5508D, TA100: 5526D, TA102: 5524D, TA1535: 5504D, TA1537:5530D) and were stored as lyophilizates in the refrigerator at 2 – 8 °C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- S9 was obtained by Trinova Biochem GmbH, Gießen.
- source of S9: livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone.
- composition of S9 mix:
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL
- concentration or volume of S9 mix in the final culture medium: 500 µL

Test concentrations with justification for top dose:

The following nominal test item concentrations were prepared for experiment 1 (Plate incorporation method):
5, 1.5, 0.5, 0.15 and 0.05 µL/plate.

The following nominal test item concentrations were prepared for experiment 2 (Pre-incubation method):
5, 2.5, 1.25, 0.63, 0.31 and 0.16 µL/plate.

The justification for the top dose is that, based on OECD 471, The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 μl/plate.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested at in a concentration of 50 mL/L in demineralized (demin.) water, dimethyl sulfoxide (DMSO), acetone and ethanol. The liquid test item is sufficiently soluble in ethanol and acetone. Based on the non-GLP pre-test, ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Dimethylsulfoxide (DMSO) was used for the positive controls 4-Nitro-1,2-phenylene diamine, benzo-a-pyrene and 2-amino-anthracene.
Demineralized water, prepared by LAUS GmbH, from an ion-exchanger, was used for for the positive controls sodium azide and mitomycin C.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Three plates with (+S9) and three plates without metabolic activation (-S9) were used per bacteria strain and concentration.
- Number of independent experiments: 2 indipendent experiments. The first was performed with the plate incorporation method. The second was performed with the pre-incubation methods.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Plate incorporation method (first experiment): The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

• 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
• 500 µL S9-mixfor test with metabolic activation or phosphate buffer for test without metabolic activation.
• 100 µL bacteria suspension.
• 2000 µL overlay agar (top agar).
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.
- Preincubation method (second experiment): The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:

• 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
• 500 µL S9-mix for test with metabolic activation or phosphate buffer for test without metabolic activation.
• 100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes at 37 C° only for the second experiment.
- Exposure duration/duration of treatment: 48 h
- Temperature: 37 °C ± 1 °C.

REFERENCES AND VALIDITY:
- Genotype Confirmation: Confirmation of genotype is performed for each batch of lyophilized bacteria by the supplier Trinova BioChem GmbH. The batches used of lyophilized bacteria met the criteria.

- Spontaneous Revertants: The number of spontaneous revertants was determined for each solvent, used in the test by investigating three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C for each strain.

- Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 10^9 cells/mL (at the least). Two replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.

- Sterility Control: Performed analogously to the test item but with solvent only and S9 (without adding bacteria) on top agar. Four replicates, incubation for 48 hours at 37 ± 1 °C.

- Solubility: Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

- Positive Controls: Using diagnostic mutagens. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate. Three replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.

EVALUATION:
Five different analysable concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.

Evaluation criteria:

A result is considered as clearly positive if all the following criteria are fulfilled:
• A concentration-related increase, in revertants
• A clear biological relevant increase in at least one concentration compared to the con-current solvent control
• At least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

EXPERIMENT 1 (PLATE INCORPORATION METHOD):
- Confirmation of the Criteria and Validity: All strains met the criterion of at least 10^9 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

- Solubility and Toxicity: In exp. 1, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of revertant col-onies was not relevantly reduced. Thus, no signs of toxicity towards the bacteria strains could be observed.

- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.

To verify this result, a further experiment (exp. 2) was performed with adapted conditions (pre-incubation method).

EXPERIMENT 2 (PRE-INCUBATION METHOD):
- Confirmation of the Criteria and Validity: All strains met the criterion of at least 10^9 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

- Solubility and Toxicity: In exp. 2, the test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was visible and not affected. The number of revertant col-onies was not relevantly reduced. Thus, no signs of toxicity towards the bacteria strains could be observed.

- Mutagenicity: No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed.

Any other information on results incl. tables

Μutagenicity Experiment I  - plate incorporation test  - Strain: S. typhimurium TA 98												Μutagenicity Experiment II  - pre-incubation test  - Strain: S. typhimurium TA 98
without metabolic activation						with metabolic activation					Conclusion	without metabolic activation						with metabolic activation					Conclusion
Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc		Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc
water	13	13	14	13±0,6	-	14	17	18	16±2,1	-	valid	water	10	12	12	11± 1,2	-	14	16	16	15± 1,2	-	valid
EtOH	12	13	14	13±1	-	13	15	19	16±3,1	-		EtOH	8	10	11	10± 1,5	-	10	12	16	13± 3,1	-
DMSO	15	15	16	15±0,6	-	16	16	19	17±1,7	-		DMSO	9	10	10	10± 0,6	-	9	13	15	13± 3,1	-
5	14	14	16	15±01,2	1,15	14	18	18	17±2,3	1,06	not mutagenic	5	8	9	12	10± 2,1	1	11	12	15	13± 2,1	1	not mutagenic
1,5	12	14	17	14±2,5	1,08	13	17	18	16±2,6	1		2,5	8	8	13	10± 2,9	1	8	11	16	12± 4	0,92
0,5	13	13	13	13±0,0	1	13	14	18	15±2,6	0,94		1,25	7	10	10	9± 1,7	0,9	11	11	15	12± 2,3	0,92
0,15	12	12	13	12±0,6	0,92	14	15	16	15±1,0	0,94		0,63	10	11	14	12± 2,1	1,2	11	12	18	14± 3,8
0,05	12	13	19	15±3,8	1,15	13	17	17	16±2,3	1		0,31	9	10	10	10± 0,6	1	10	16	17	14± 3,8	1,08
NDP/BaP	556	560	564	560±4	37,33	80	94	95	90±8,4	5,29	valid	NDP/BaP	512	560	576	549± 33,3	54,9	92	96	104	97± 6,1	8,08
Μutagenicity Experiment I  - plate incorporation   - Strain: S. typhimurium TA 100												Μutagenicity Experiment II  - pre-incubation test   - Strain: S. typhimurium TA 100
without metabolic activation						with metabolic activation					Conclusion	without metabolic activation						with metabolic activation					Conclusion
Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc		Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc
water	60	65	69	65± 4,5	-	60	66	68	65± 4,2	-	valid	water	52	67	70	63± 9,6	-	56	60	62	59± 3,1	-	valid
EtOH	53	59	66	59± 6,5	-	56	56	60	57± 2,3	-		EtOH	48	56	60	55± 6,1	-	53	56	57	55± 2,1	-
DMSO	60	66	70	65± 5	-	64	68	70	67± 3,1	-		DMSO	52	55	69	59± 9,1	-	53	54	70	59± 9,5	-
5	66	70	73	70± 3,5	1,19	54	57	58	56± 2,1	0,98	not mutagenic	5	43	56	57	52± 7,8	0,95	46	54	59	53± 6,6	0,96	not mutagenic
1,5	56	61	68	62± 6	1,05	53	56	57	55± 2,1	0,96		2,5	47	52	58	52± 5,5	0,95	47	50	60	52± 6,8	0,95
0,5	56	58	62	59± 3,1	1	60	61	67	63± 3,8	1,11		1,25	47	49	53	50± 3,1	0,91	50	55	57	54± 3,6	0,98
0,15	58	58	60	59± 1,2	1	57	61	64	61± 3,5	1,07		0,63	41	43	50	45± 4,7	0,82	45	47	53	48± 4,2	0,87
0,05	65	66	68	66± 1,5	1,12	56	60	62	59± 3,1	1,04		0,31	47	49	54	50± 3,6	0,91	50	54	56	53± 3,1	0,96
Na-azide/2-AA	496	536	568	533±36,1	8,2	1112	1168	1208	1163± 48,2	17,36	valid	Na-azide/2-AA	400	448	536	461± 69	7,32	1176	1208	1240	1208± 32	20,47	valid
Μutagenicity Experiment I  - plate incorporation test   - Strain: S. typhimurium TA 102												Μutagenicity Experiment II  - pre-incubation test   - Strain: S. typhimurium TA 102
without metabolic activation						with metabolic activation					Conclusion	without metabolic activation						with metabolic activation					Conclusion
Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc		Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc
water	148	156	160	155± 6,1	-	144	144	148	145± 2,3	-	valid	water	152	156	164	157± 6,1	-	148	164	164	159± 9,2	-	valid
EtOH	140	152	160	151± 10,1	-	136	144	156	145± 10,1	-		EtOH	140	156	160	152± 10,6	-	156	172	176	168± 10,6	-
DMSO	144	152	164	153± 10,1	-	140	160	160	153± 11,5	-		DMSO	140	152	168	153± 14	-	148	156	164	156± 8	-
5	148	156	160	155± 6,1	1.03	148	152	164	155± 8,3	1.07	not mutagenic	5	144	148	164	152± 10,6	1	136	144	160	147± 12,2	0,88	not mutagenic
1,5	144	148	160	151± 8,3	1.00	140	148	152	147± 6,3	1.01		2,5	144	148	156	149± 6,1	0,98	160	164	164	163± 2,3	0,97
0,5	136	136	140	137± 2,3	0.91	132	136	144	137± 6,1	0.94		1,25	144	152	164	153± 10,1	1,01	148	160	164	157± 8,3	0,93
0,15	132	144	156	144± 12	0.95	132	132	136	133± 2,3	0.92		0,63	144	156	164	155± 10,1	1,02	160	168	172	167± 6,1	0,99
0,05	132	140	160	144± 14,4	0.95	128	136	156	140± 14,4	0.97		0,31	144	148	160	151± 8,3	0,99	164	172	172	169± 4,6	1,01
MMC/2-AA	480	484	528	497±26,6	3,34	472	484	500	485±14,0	3,17	valid	MMC/2-AA	808	872	904	861±48,9	5,48	1016	1048	1120	1061±53,3	6,8	valid
Μutagenicity Experiment I  - plate incorporation test   - Strain: S. typhimurium TA 1535												Μutagenicity Experiment II  - pre-incubation test   - Strain: S. typhimurium TA 1535
without metabolic activation						with metabolic activation					Conclusion	without metabolic activation						with metabolic activation					Conclusion
Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc		Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc
water	7	8	8	8± 0,6	-	8	8	10	9± 1,2	-	valid	water	7	9	9	8± 1,2	-	6	6	7	6± 0,6	-	valid
EtOH	7	8	8	8± 0,6	-	9	9	11	10± 1,2	-		EtOH	5	6	6	6± 0,6	-	8	8	8	8± 0,00	-
DMSO	7	7	7	7± 0,0	-	7	8	9	8± 1,0	-		DMSO	6	7	8	7± 1	-	6	7	9	7± 1,5	-
5	7	8	9	8± 1,0	1	7	8	9	8± 1,0	0,8	not mutagenic	5	5	6	8	6± 1,5	1	6	7	7	7± 0,6	0,88	not mutagenic
1,5	8	9	10	9± 1	1,13	7	9	11	9± 2	0,9		2,5	5	6	7	6± 1	1	6	7	10	8± 2,1	1
0,5	8	8	9	8± 0,6	1	7	7	8	7± 0,6	0,7		1,25	7	8	8	8± 0,6	1,33	7	8	9	8± 1	1
0,15	8	9	10	9± 1	1,13	9	9	11	10± 1,2	1		0,63	6	7	8	7± 1	1,17	7	7	9	8± 1,2	1
0,05	8	9	9	9± 0,6	1,13	8	9	11	9± 1,5	0,9		0,31	7	8	9	8± 1	1,33	6	6	7	6± 0,6	0,75
Na-azide/2-AA	184	152	190	175±20,4	21,88	88	111	114	104± 14,2	13	valid	Na-azide/2-AA	132	136	158	142±14,0	17,75	120	148	160	143± 20,5	20,43	valid
Μutagenicity Experiment I  - plate incorporation test   - Strain: S. typhimurium TA 1537												Μutagenicity Experiment II  - pre-incubation test   - Strain: S. typhimurium TA 1537
without metabolic activation						with metabolic activation					Conclusion	without metabolic activation						with metabolic activation					Conclusion
Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc		Test item (μL per plate)	Revertants per plate			mean ± sd	Rt/Rc	Revertants per plate			mean ± sd	Rt/Rc
water	3	4	6	4± 1,5	-	4	4	5	4± 0,6	-	valid	water	3	3	5	4± 1,2	-	5	5	6	5± 0,6	-	valid
EtOH	3	3	5	4± 1,2	-	3	5	6	5± 1,5	-		EtOH	2	3	4	3± 1	-	3	4	6	4± 1,5	-
DMSO	3	5	7	5± 2	-	4	5	6	5± 1	-		DMSO	4	5	5	5± 0,6	-	3	3	6	4± 1,7	-
5	4	5	6	5± 1	1,25	3	5	5	4± 1,2	0,8	not mutagenic	5	3	3	3	3± 0,00	1	3	4	5	4± 1	1	not mutagenic
1,5	3	4	6	4± 1,5	1	3	4	8	5± 2,6	1		2,5	2	3	4	3± 1	1	4	5	6	5± 1	1,25
0,5	3	4	8	5± 2,6	1,25	4	4	4	4± 0	0,8		1,25	3	3	3	3± 0,00	1	2	3	4	3± 1	0,75
0,15	3	4	7	5± 2,1	1,25	5	6	6	6± 0,6	1,2		0,63	3	3	3	3± 0,00	1	4	4	5	4± 0,6	1
0,05	3	3	8	5± 2,9	1,25	3	6	7	5± 2,1	1		0,31	3	3	3	3± 0,00	1	3	4	4	4± 0,6	1
NDP/2-AA	52	61	68	60±8	12	81	89	91	87±5,3	17,4	valid	NDP/2-AA	65	73	83	74±9	14,8	50	54	61	55±5,6	13,75	valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Di(tridecyl) 3,3'-thiodipropionate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions of this study.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of Di(tridecyl) 3,3'-thiodipropionate in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) based on the most recent Guidelines OECD 471 (2020) and EU Method B.13/14 (2008). The test was performed in two valid experiments at a cell density of 10^9 cells/mL in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Therefore, a further experiment with adapted conditions (pre-incubation method) was performed (exp. 2).

Based on the results of the first experiment, the test item was tested up to concentrations of 5 µL/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method. The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.