Methyl toluene-4-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity in vitro - Bacterial Reverse Mutation Assay

Mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escerichia coli strain WP2 uvrA with and without metabolic activation.

Genetic Toxicity in vitro - HPRT assay

CHO K1 Chinese hamster ovary cells at the hprt locus with and without metabolic activation (±S9-mix)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 to 15 June 2017

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:

OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008, B.13/14. „Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

histidine (his) and tryptophan (trp)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).
In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test): 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Examined concentrations in the Initial Mutation Test were 5000, 1581, 1000, 500, 250, 158.1 and 100 μg/plate and examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 1000, 500, 250, 158.1, 100 and 50 μg/plate.

Vehicle / solvent:

The following chemicals were used for vehicle (solvent) control groups:

Distilled water:
Manufacturer: Hungaro-Gal Kft.
Batch No.: 802 0117
Expiry date: 23 July 2017

Dimethyl sulfoxide (DMSO)*:
Supplier: Sigma-Aldrich Co.
Batch No.: STBG8411
Expiry date: 29 February 2020

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used, in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation the plate incorporation method was used; in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains with and without metabolic activation the pre-incubation method was used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test item was insoluble at 100 mg/mL concentration using Distilled water. The test item was soluble at this concentration using DMF and DMSO. Due to the better biocompatibility DMSO was selected as vehicle (solvent) for the study. The test item was formulated in the selected vehicle (solvent). The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 1000, 500, 250, 158.1 and 100 μg/plate and examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 1000, 500, 250, 158.1, 100 and 50 μg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test and in the Confirmatory Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test and as a Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item solution (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains with and without metabolic activation, since in the Initial Mutation Test positive effect was observed in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation, the plate incorporation method was used.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hour.

Rationale for test conditions:

The experimental methods were conducted according to the methods described Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced/slightly reduced background lawn 1581 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced/slightly reduced background lawn 1581 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced/slightly reduced background lawn 1581 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced/slightly reduced background lawn 1581 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced/slightly reduced background lawn 1581 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CONCENTRATION RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
Preliminary Concentration Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the Preliminary Concentration Range Finding Test, a dose–related pattern was observed in the number of revertant colonies in Salmonella typhimurium TA100 strain with and without metabolic activation. The calculated mutation factor values were over the biologically relevant threshold values 2 (except at 10 μg/plate).
No precipitate of the test item was detected in the Preliminary Concentration Range Finding Test in all examined strains with and without metabolic activation.
Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in all examined strains with and without metabolic activation on the plates at 5000 and 2500 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TEST
In the Initial Mutation Test, the plate incorporation method was used, in the Confirmatory Mutation Test the plate incorporation method or the pre-incubation method were used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 1000, 500, 250, 158.1 and 100 μg/plate and the examined test concentrations in the Confirmatory Mutation Test were 5000, 1581, 1000, 500, 250, 158.1, 100 and 50 μg/plate
In the Initial Mutation Test using the plate incorporation method, a clear positive effect of the test item was obtained in Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 2 at several concentrations and dose dependence was also observed.
The observed positive effect was confirmed in the Confirmatory Mutation Test using the plate incorporation method in Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation. This result demonstrated the positive effect was reproducible and showed dose dependent pattern.
A positive effect of the test item was obtained in the Confirmatory Mutation Test using the pre-incubation method in Salmonella typhimurium TA1535 strain as the calculated mutation factor values were over the biologically relevant threshold value of 2 at 500 μg/plate concentration without metabolic activation and 500, 250 and 158.1 μg/plate concentrations with metabolic activation and dose dependent pattern was also observed. However, in the Initial Mutation Test this result was not observed. In the Initial Mutation Test no positive effect was observed in this strain, so the effect was not fully reproducible.
No precipitate was observed in the main tests in all examined strains with and without metabolic activation.
Reduced/slightly reduced background lawn was observed in the Initial Mutation Test in all examined strains with and without metabolic activation on the plates at 5000 and in Salmonella typhimurium TA100 strain at 1581 μg/plate.
Reduced/slightly reduced background lawn was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA1537 strains with metabolic activation on the plates at 5000, 1581, 1000 μg/plate and without metabolic activation at 5000, 1581, 1000, 500, 250 μg/plate and in Salmonella typhimurium TA1535 strains with and without metabolic activation on the plates at 5000, 1581, 1000 μg/plate. The same effect was detected in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5000 μg/plate concentration and in Salmonella typhimurium TA100 strain with and without metabolic activation at 5000 and 1581 μg/plate concentrations.
Taking into account the results at all concentrations under all conditions for each bacterial strain, the results for Salmonella typhimurium TA98, TA1537 with and without metabolic activation were negative in the Initial Mutation Test*. The results for Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation using the plate incorporation method were reproducibly positive. Positive results were observed using the pre-incubation method in Salmonella typhimurium TA1535 with and without metabolic activation. The results for Salmonella typhimurium TA1535 did not show a fully reproducible positive result, the pattern of results indicates the result for this strain is equivocal. (further experiment is not considered to be required because two bacterial strains results are clearly positive).
*Note: In the Confirmatory Mutation Test cytotoxicity was seen in Salmonella typhimurium TA98 and TA1537 bacterial strains without metabolic activation, with less than 5 concentrations for evaluation, but since the test item is already classified as positive, there is no requirement for further testing.

Summary Table of the Preliminary Concentrations Range Finding Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	23.3	23.3	104.7	125.3
	MF	1.17	0.97	1.08	1.06
DMSO control	Mean	20.0	24.0	97.3	118.7
	MF	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	104.7	--
	MF	--	--	1.08	--
5000	Mean	0.3	0.3	5.3	10.0
	MF	0.02	0.01	0.05	0.08
2500	Mean	3.0	11.3	13.3	109.0
	MF	0.15	0.47	0.14	0.92
1000	Mean	18.7	34.0	925.3	1210.7
	MF	0.93	1.42	9.51	10.20
316	Mean	21.3	27.3	1004.7	1197.3
	MF	1.07	1.14	10.32	10.09
100	Mean	17.3	28.0	461.7	512.0
	MF	0.87	1.17	4.74	4.31
31.6	Mean	19.3	24.3	239.3	339.7
	MF	0.97	1.01	2.46	2.86
10	Mean	13.0	28.7	148.7	177.0
	MF	0.65	1.19	1.53	1.49
NPD (4μg)	Mean	388.0	--	--	--
	MF	19.40	--	--	--
2AA (2μg)	Mean	--	2368.7	--	2384.0
	MF	--	98.69	--	20.09
SAZ (2μg)	Mean	--	--	1192.0	--
	MF	--	--	11.39	--

 

Summary Table of the Initial Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.7	26.3	116.3	130.0	9.3	13.7	13.7	15.7	66.3	62.7
	MF	1.15	0.88	1.00	1.03	0.61	1.03	1.00	1.15	0.96	0.90
DMSO control	Mean	19.7	30.0	116.3	126.0	15.3	13.3	13.7	13.7	69.3	69.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	114.7	--	13.0	--	--	--	64.3	--
	MF	--	--	0.99	--	0.85	--	--	--	0.93	--
5000	Mean	9.7	4.0	55.7	183.3	5.3	9.7	4.7	9.3	167.7	199.7
	MF	0.19	0.13	0.48	1.46	0.35	0.73	0.34	0.68	2.42	2.88
1581	Mean	18.0	26.7	165.0	329.3	18.3	16.0	10.7	11.7	422.7	622.7
	MF	0.92	0.89	1.42	2.61	1.20	1.20	0.78	0.85	6.10	8.98
1000	Mean	21.0	24.0	425.3	997.3	16.7	17.3	11.3	14.3	457.3	492.0
	MF	1.07	0.80	3.66	7.92	1.09	1.30	0.83	1.05	6.60	7.10
500	Mean	27.3	33.0	981.3	1364.0	17.0	14.7	13.3	13.3	382.7	441.3
	MF	1.39	1.10	8.44	10.83	1.11	1.10	0.98	0.98	5.52	6.37
250	Mean	26.0	31.0	865.4	1302.7	14.7	16.0	11.0	13.7	354.7	460.7
	MF	1.32	1.03	7.44	10.34	0.96	1.20	0.80	1.00	5.12	6.64
158.1	Mean	21.0	30.3	708.0	926.0	14.7	14.0	10.0	11.3	277.0	321.3
	MF	1.07	1.01	6.09	7.35	0.96	1.05	0.73	0.83	4.00	4.63
100	Mean	20.0	25.3	565.3	707.7	13.3	13.3	9.7	11.3	219.3	226.7
	MF	1.02	0.84	4.86	5.62	0.87	1.00	0.74	0.83	3.16	3.25
NPD (4μg)	Mean	424.0	--	--	--	--	--	--	--	--	--
	MF	21.56	--	--	--	--	--	--	--	--	--
2AA (2μg)	Mean	--	2473.3	--	2408.0	--	206.3	--	211.7	--	--
	MF	--	82.44	--	19.11	--	15.48	--	15.49	--	--
2AA (50μg)	Mean	--	--	--	--	--	--	--	--	--	246.7
	MF	--	--	--	--	--	--	--	--	--	3.56
SAZ (2μg)	Mean	--	--	1078.7	--	1061.3	--	--	--	--	--
	MF	--	--	9.41	--	91.64	--	--	--	--	--
9AA (50μg)	Mean	--	--	--	--	--	--	457.3	--	--	--
	MF	--	--	--	--	--	--	33.46	--	--	--
MMS (2μL)	Mean	--	--	--	--	--	--	--	--	953.3	--
	MF	--	--	--	--	--	--	--	--	14.82	--

 

Summary Table of the Confirmatory Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	20.0	35.0	137.0	129.3	1.0	11.0	15.3	18.7	61.0	68.3
	MF	0.63	1.01	1.08	1.11	1.25	1.10	0.84	0.95	1.02	1.22
DMSO control	Mean	31.7	34.7	126.7	116.7	8.0	10.0	18.3	19.7	60.0	56.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	125.0	--	12.3	--	--	--	63.0	--
	MF	--	--	0.99	--	1.54	--	--	--	1.05	--
5000	Mean	3.3	0.3	30.3	59.3	8.7	1.7	0.0	0.0	137.7	253.3
	MF	0.11	0.01	0.24	0.51	1.08	0.17	0.00	0.00	2.29	4.20
1581	Mean	2.0	4.3	230.0	300.7	6.3	6.0	2.3	3.7	417.3	618.7
	MF	0.06	0.13	1.82	2.58	0.79	0.60	0.13	0.18	6.96	11.05
1000	Mean	8.0	26.7	646.7	1016.0	6.7	16.7	7.0	9.3	418.7	618.7
	MF	0.25	0.77	5.11	8.71	0.83	1.67	0.38	0.47	6.98	9.10
500	Mean	26.3	44.0	944.0	1236.0	18.7	22.3	13.7	16.0	406.7	405.3
	MF	0.83	1.27	7.45	10.59	2.33	2.23	0.75	0.81	6.78	7.24
250	Mean	9.7	33.0	845.3	1242.7	12.0	28.7	3.7	16.3	388.7	445.3
	MF	0.31	0.95	6.67	10.65	1.50	2.87	0.20	0.83	6.48	7.95
158.1	Mean	29.0	25.7	694.7	864.0	15.0	23.3	19.3	18.0	302.3	322.0
	MF	0.92	0.74	5.48	7.41	1.88	2.33	1.05	0.92	5.04	5.75
100	Mean	28.0	31.7	468.0	496.7	13.7	17.3	15.0	18.3	231.7	240.7
	MF	0.88	0.91	3.69	4.26	1.71	1.73	0.82	0.93	3.86	4.30
50	Mean	26.3	30.0	329.3	405.0	10.0	12.7	17.0	16.3	148.3	169.0
	MF	0.83	0.87	2.60	3.47	1.25	1.28	0.93	0.83	2.47	3.02
NPD (4μg)	Mean	489.3	--	--	--	--	--	--	--	--	--
	MF	15.45	--	--	--	--	--	--	--	--	--
2AA (2μg)	Mean	--	2312.0	--	2226.7	--	262.7	--	244.3	--	--
	MF	--	66.69	--	19.09	--	26.27	--	12.42	--	--
2AA (50μg)	Mean	--	--	--	--	--	--	--	--	--	284.7
	MF	--	--	--	--	--	--	--	--	--	5.08
SAZ (2μg)	Mean	--	--	1090.7	--	1157.3	--	--	--	--	--
	MF	--	--	8.73	--	93.84	--	--	--	--	--
9AA (50μg)	Mean	--	--	--	--	--	--	490.7	--	--	--
	MF	--	--	--	--	--	--	26.76	--	--	--
MMS (2μL)	Mean	--	--	--	--	--	--	--	--	1033.3	--
	MF	--	--	--	--	--	--	--	--	16.40	--

 

Historical Control Data

Untreated control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.7	103.6	11.8	7.2	33.4	29.7	111.7	11.5	8.9	39.1
St. dev.	5.8	21.4	5.1	3.3	9.7	6.8	19.6	3.9	3.8	9.9
Range	9-50	54-210	1-46	1-24	11-82	10-56	65-204	1-39	1-29	16-89
n	1371	1357	1365	1371	1374	1377	1365	1373	1380	1371
DMSO control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1536	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.7	98.9	12.0	7.1	32.3	28.7	109.5	11.3	8.7	38.1
St. dev.	5.7	20.7	5.0	3.3	9.6	7.0	20.7	3.8	3.7	9.7
Range	6-55	40-217	1-43	1-25	7-81	11-67	53-229	2-33	1-29	9-85
n	1482	1473	1479	1485	1482	1487	1476	1487	1491	1482
Distilled water control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E.coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	23.5	103.2	11.9	7.8	34.5	30.8	112.2	11.3	9.3	40.3
St. dev.	6.0	22.4	4.9	3.4	9.8	7.1	21.8	3.7	3.7	10.0
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-24	13-91
n	267	1359	1365	270	1392	267	1371	1380	267	1383
DMF control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.4	89.9	11.2	6.9	34.7	28.1	100.3	11.0	8.0	38.0
St. dev.	5.6	17.8	4.7	3.1	12.3	7.0	19.2	3.6	3.1	10.2
Range	8-38	54-152	1-34	1-19	16-99	13-49	60-156	3-21	1-23	17-76
n	216	216	216	216	207	216	216	216	213	207
Acetone control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TS1535	TA1537	E.coli	TA98	TA100	TA5135	TA1537	E. coli
Mean	22.6	98.1	12.1	7.4	35.0	29.1	108.1	11.1	8.6	40.5
St. dev.	5.1	15.4	5.8	2.9	9.3	6.7	14.2	3.4	3.3	9.0
Range	11-39	62-160	4-49	1-17	17-62	15-52	66-177	4-22	1-19	17-69
n	278	279	279	279	276	279	279	282	279	279
Positive reference control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	357.2	1229.3	1169.8	454.1	1034.3	2410.2	2429.6	235.1	221.3	257.4
St. dev.	113.8	207.5	204.2	169.7	141.7	317.7	291.6	135.9	56.2	113.4
Range	152-2336	536-2120	208-2440	149-2104	488-1708	312-4918	1192-5240	101-2216	117-838	125-2512
n	1371	1359	1365	1371	1377	1378	1365	1377	1380	1371

TA98: Salmonella typhimurium TA98; TA100: Salmonella typhimurium TA100; TA1535: Salmonella typhimurium TA1535; TA1537: Salmonella typhimurium TA1537; E.coli: Escherichia coli WP2 uvrA; n: number of cases

Conclusions:

The reported data of the mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item PTSM (Batch Number: 609271) was shown to be mutagenic under the test conditions used in this study.

Executive summary:

The test item PTSM was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and Confirmatory Mutation Test (in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation the Plate Incorporation Method was used; in the case of Salmonella typhimurium TA98, TA1535, TA1537 strains with and without metabolic activation the Pre-Incubation Method was used).

 

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Preliminary Concentration Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) were 5000, 1581, 1000, 500, 250, 158.1 and 100 μg/plate and in the Confirmatory Mutation Test (5 strains) were 5000, 1581, 1000,

500, 250, 158.1, 100 and 50 μg/plate.

 

In the Preliminary Concentration Range Finding Test in the case of Salmonella typhimurium TA100 strain with and without metabolic activation, in the Initial Mutation Test and Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation, a clear, reproducible positive effect was obtained using the plate incorporation method. In the Confirmatory Mutation Test a positive effect was obtained in Salmonella typhimurium TA1535 bacterial strains with and without metabolic activation using the pre-incubation method, however this result was not observed in the first assay.

 

No precipitate was observed in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in all examined strains with and without metabolic activation on the plates at 5000 and 2500 μg/plate.

The same effect was detected in the Initial Mutation Test in all examined strains with and without metabolic activation at higher concentrations (5000 and/or 1581 μg/plate).

Reduced/slightly reduced background lawn was observed in the Confirmatory Mutation Test in in all examined strains with and without metabolic activation at several concentrations.

 

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable* concentrations were presented in all strains of the main tests. The tests were considered to be valid.

 

*Note: In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in Salmonella typhimurium TA98 and TA1537 bacterial strains without metabolic activation.

However, in the Initial Mutation Test and in the Confirmatory Mutation Test clearly positive results were observed in Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA strains with and without metabolic activation and in the Confirmatory Mutation Test positive results were observed in Salmonella typhimurium TA1535 strain with and without metabolic activation.

 

Since the overall conclusion of this study is clearly Positive, no additional Complementary Confirmatory Test is required because the results are fully sufficient to make a regulatory Positive conclusion. Cytotoxicity in Salmonella typhimurium TA98 and TA1537 bacterial strains was seen without metabolic activation, with less than 5 concentrations for evaluation, but since the test item is already classified as positive, there is no requirement for further testing.

 

The reported data of the mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item PTSM (Batch Number: 609271) was shown to be mutagenic under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May to 25 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

CHO K1 Chinese hamster ovary cells at the hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

hprt

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Male Wistar rats treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days
- method of preparation of S9 mix: The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 minutes at 9000 g, and then the supernatant was decanted and retained.
- concentration or volume of S9 mix and S9 in the final culture medium : For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.

Test concentrations with justification for top dose:

Assay 1
For the 5-hour treatment in the presence of S9-mix, the following concentrations were examined: 120, 60, 50, 40, 30, 15. 7.5 and 3.75 μg/mL
For the 5-hour treatment in the absence of S9-mix, the following concentrations were examined: 120, 60, 50, 40, 30, 15. 7.5 and 3.75 μg/mL

Assay 2
For the 5-hour treatment in the presence of S9-mix, the following concentrations were examined: 120, 60, 50, 40, 30, 15. 7.5, 3.75 and 1.875 μg/mL
For 24-hour treatment in the absence of S9-mix, the following concentrations were examined: 120, 60, 50, 40, 30, 15. 7.5, 3.75 and 1.875 μg/mL

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Dose levels above 40 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Mutagenic effect of PTSM was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.

Executive summary:

An in vitro mammalian cell assay [1-2] was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of PTSM to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).

Dimethyl sulfoxide (DMSO) was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test.

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least four evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid.

In conclusion, mutagenic effect of PTSM was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic Toxicity in vitro - Bacterial Reverse Mutation Assay

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Preliminary Concentration Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) were 5000, 1581, 1000, 500, 250, 158.1 and 100 μg/plate and in the Confirmatory Mutation Test (5 strains) were 5000, 1581, 1000,

500, 250, 158.1, 100 and 50 μg/plate.

 

In the Preliminary Concentration Range Finding Test in the case of Salmonella typhimurium TA100 strain with and without metabolic activation, in the Initial Mutation Test and Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation, a clear, reproducible positive effect was obtained using the plate incorporation method. In the Confirmatory Mutation Test a positive effect was obtained in Salmonella typhimurium TA1535 bacterial strains with and without metabolic activation using the pre-incubation method, however this result was not observed in the first assay.

No precipitate was observed in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in all examined strains with and without metabolic activation on the plates at 5000 and 2500 μg/plate.

The same effect was detected in the Initial Mutation Test in all examined strains with and without metabolic activation at higher concentrations (5000 and/or 1581 μg/plate).

Reduced/slightly reduced background lawn was observed in the Confirmatory Mutation Test in in all examined strains with and without metabolic activation at several concentrations.

 

The reported data of the mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item PTSM (Batch Number: 609271) was shown to be mutagenic under the test conditions used in this study.