Dipotassium methanedisulphonate

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:

8 November 2017 - 20 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Dipotassium methanedisulphonate

EC Number:

228-543-0

EC Name:

Dipotassium methanedisulphonate

Cas Number:

6291-65-2

Molecular formula:

CH4O6S2.2K

IUPAC Name:

dipotassium methanedisulphonate

Test material form:

solid

Method

Target gene:

Histidine-requiring gene in Salmonella thyphimurium

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

10% v/v S9 mix from rat liver

Test concentrations with justification for top dose:

156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.
In a previous solubility test, precipitation of the test item was not observed at the tested concentration of 5000 µg/plate thus it was selected as the highest concentration to be tested for the initial toxicity-mutation test. Results from this initial toxicity test revealed that there was no positive mutagenic effect up to the tested concentration of 5000 µg/plate, thus this concentration was finally selected as the highest concentration to be tested in the confirmatory mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: water is recommended by OECD TG 471 and the test item was completely soluble in distilled water at 25000 µg/mL as stated in the solubility test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene (10 μg/plate for TA1537, TA1535 and TA102, and 5.0 µg/plate for TA98 and TA100 with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1–2 x10^9 bacteria/mL

DURATION
- Preincubation period: Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to early stationary or late exponential phase.
- Exposure duration: 48 h incubation period at 37± 1

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies.

Rationale for test conditions:

According to OECD TG 471

Evaluation criteria:

A result was considered positive if concentration-related increase over the range tested and/or a reproducible increased at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Strains TA1535, TA1537: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strain TA98, TA100 and TA102: Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
A response should meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) to be evaluated as positive.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

Key result

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:

valid

Positive controls validity:

valid

Test results 2

Key result

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:

valid

Positive controls validity:

valid

Test results 3

Key result

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:

valid

Positive controls validity:

valid

Test results 4

Key result

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:

valid

Positive controls validity:

valid

Test results 5

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: A first stock solution up to 50000 µg/mL was prepared for the highest test concentration of 5000 µg/plate.
- Precipitation: No precipitation of the test item was observed at any concentration or tester strain used.
- Definition of acceptable cells for analysis:
All tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation. All the tester strains demonstrated the requirement of the biotin except strain TA102 which is biotin independent. All the tester strains showed sensitivity to UV exposure except TA102 which is wild type. All the tester strains demonstrated requirement of histidine for their growth. Tester strains TA98, TA100 and TA102 exhibited resistance to ampicillin, demonstrating presence of the pKM101 plasmid. Tester strains TA102 exhibited resistance to tetracycline, demonstrating presence of the pAQ1 plasmid.
Cell densities (OD at 660 nm) of all tester strain were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.

RANGE-FINDING/SCREENING STUDIES:
Before commencing the confirmatory mutation test (main test), test item was tested for initial toxicity-mutation test using all five tester strains of Salmonella typhimurium. The experiment was conducted for test concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate of test item, both in the absence and presence of metabolic activation system (5% v/v S9 mix). Results revealed that there was no positive mutagenic effect in any tester strains up to the highest dose tested. Hence, 5000 µg/plate of test item was selected as the highest concentration to be tested in the confirmatory mutation test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 3 in "any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: see table 3 in "any other information on results incl. tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies. Normal background lawn pattern without reduction in revertant colonies was observed up to the highest dose level of 5000 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester stains.

Any other information on results incl. tables

Table 1: Mean Count of His+ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Test)

Concentration of Test Item (µg/plate)	His^+Revertant Colonies/Plate [Absence of Metabolic Activation]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	8.00	3.00	14.67	2.08	19.00	3.00	133.33	8.02	233.33	9.61
156.25	8.67	3.51	15.67	4.51	17.33	2.52	134.67	7.57	232.67	8.02
312.5	8.33	2.52	14.33	5.51	18.67	5.51	132.67	4.93	234.00	7.55
625	5.33	2.52	15.67	2.08	18.67	4.04	131.67	11.02	235.33	8.08
1250	8.00	2.65	15.67	3.51	19.33	2.08	135.67	7.09	230.33	10.07
2500	9.33	3.79	13.67	3.06	19.67	7.64	133.67	9.29	232.67	7.64
5000	8.00	1.00	15.67	4.51	19.33	6.11	133.33	10.21	233.33	6.66
PC	228.00	78.25	378.00	113.42	552.67	245.50	932.67	58.23	961.33	98.59
2Aa	-	-	-	-	-	-	131.67	8.02	-	-

Keys: SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not Applicable, Test Item = Dipotassium methanedisulphonate.

Table 2: Mean Count of His+ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Test)

Concentration of Test Item (µg/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	8.00	2.65	15.67	4.04	20.67	4.04	137.00	12.12	236.00	12.00
156.25	7.00	3.00	15.33	2.31	20.33	4.73	135.33	18.58	238.33	6.11
312.5	9.33	2.52	16.00	3.00	21.00	5.29	138.33	7.51	232.67	10.69
625	8.00	3.61	14.33	2.52	20.33	2.52	135.33	6.66	236.33	11.59
1250	8.00	2.65	16.67	5.13	21.33	4.04	137.00	13.11	235.00	12.29
2500	8.67	4.16	15.00	4.00	21.00	4.58	136.67	15.53	235.67	5.51
5000	9.33	2.08	16.67	5.03	21.33	4.73	138.67	15.31	236.67	7.77
PC-2Aa	310.67	164.89	496.00	157.55	785.33	89.19	858.00	40.58	964.00	152.56

Keys: SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive Control, 2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), Test Item = Dipotassium methanedisulphonate.

Table 3: Historical Control Data (Data of 112 studies conducted at JRF during July 2016 to July 2017)

Negative Control (Distilled Water) without S9 Mix
Strain	TA1537	TA1535	TA98	TA100	TA102	E. coliWP2uvrA	E. coliWP2uvrA (pKM101)
Mean Revertants per Plate	8.50	18.46	22.73	130.74	229.33	28.80	119.75
Standard Deviation	2.87	4.65	5.00	11.15	11.69	4.09	7.76
Maximum	15	29	33	161	269	33	135
Minimum	2	9	10	103	203	24	108
Negative Control (Distilled Water) with S9 Mix
Mean Revertants per Plate	9.32	20.08	24.13	133.63	233.14	30.00	123.80
Standard Deviation	2.91	4.87	5.47	10.66	12.22	5.24	8.90
Maximum	16	33	36	157	268	37	143
Minimum	3	10	13	105	205	25	112
Negative Control (Dimethyl Sulfoxide) without S9 Mix
Mean Revertants per Plate	8.40	18.37	22.79	131.37	228.89	27.33	115.16
Standard Deviation	2.73	4.44	4.92	10.65	12.48	4.86	7.68
Maximum	13	28	34	155	277	35	131
Minimum	3	8	13	107	202	20	100
Negative Control (Dimethyl Sulfoxide) with S9 Mix
Mean Revertants per Plate	8.96	20.03	24.24	134.50	232.55	29.67	119.24
Standard Deviation	3.02	4.97	5.43	10.53	13.12	5.81	8.04
Maximum	15	31	36	159	266	40	136
Minimum	3	8	12	112	156	22	103
Positive Control without S9 Mix
Mean Revertants per Plate	274.27	376.70	542.14	880.92	1058.59	461.35	925.09
Standard Deviation	94.89	113.61	148.27	175.36	152.48	143.11	195.70
Maximum	1015	836	1062	1333	1543	794	1235
Minimum	75	134	217	417	427	281	417
Positive Control with S9 Mix
Mean Revertants per Plate	300.52	419.02	671.47	968.94	1088.70	538.45	993.91
Standard Deviation	98.11	122.05	171.10	158.04	154.86	126.44	220.14
Maximum	682	802	1463	1441	1581	753	1289
Minimum	73	191	283	577	586	238	389

Negative control:

-Distilled Water

Positive controls in the absence of metabolic activation:

-TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate).

Positive controls in the presence of metabolic activation:

-2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test substance is non-mutagenic to any of the five strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100 and TA102).

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and TA102 in the presence and absence of metabolic activation (S9 mix prepared from the livers of rats). The test item was dissolved in distilled water, based on its solubility. In the initial toxicity-mutation test the bacterial cultures were exposed to test item at 8 concentrations (two plates/concentration) between 1.5 and 5000 µg/plate . Normal background lawn pattern was observed up to the highest dose in all tester strains, no mutagenic effect was observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. In the main test, the cultures were exposed to test item at 6 concentrations (three plates/concentration) between 156.25 and 5000 µg/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies. All validity criteria were met. No positive effect was obtained in the examined bacterial strains with and without metabolic activation system.