2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonic acid-potassium (1/1)

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:

2018-06-14 to 2018-08-21

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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:

potassium 2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethane-1-sulfonate

EC Number:

618-797-4

Cas Number:

91893-72-0

Molecular formula:

C10H9NO5S.K

IUPAC Name:

potassium 2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethane-1-sulfonate

Test material form:

solid: particulate/powder

Method

Target gene:

histidine locus

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:

S9-Mix (rat liver)

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160, 5000 µg/plate; the results of a preliminary test revealed no cytotoxicity up to the top dose of 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: highly purified water
- Justification for choice of solvent/vehicle: Solubility of the test item

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): 1E+08 - 1E+09 cells/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 per concentration and/or control in 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants by at least 50%, a clearing or dimunition of the background lawn or by the degree of survival of the treated cultures

Rationale for test conditions:

As recommended by the test guideline

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

U-test according to MANN and WHITNEY and the Spearman's rank correlation coefficient

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: 93 g/L
- Precipitation: Not reported

RANGE-FINDING/SCREENING STUDIES: A preliminary (plate incorporation test, without and with metabolic activation) with the tester strain TA100 revealed not cytotoxicity up to the top dose of 5000 µg/plate


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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in the number of spontaneous revertants by at least 50%, a clearing or dimunition of the background lawn or by the degree of survival of the treated cultures

Any other information on results incl. tables

 Tables 1 & 2: History profile of Negative and Positive Control Values of three years (n=88), Data obtained from plate incorporation and preincubation tests

Negative Reference item

Strain	TA98		TA100		TA102		TA1535		TA1537
S9-Mix	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	30.2	31.9	145.9	145.1	278.1	279.0	19.7	19.9	6.7	6.7
SD	5.6	5.9	19.6	19.9	16.9	17.5	4.4	4.6	1.8	1.8
Min	20	20	95	100	245	203	10	10	2	2
Max	49	49	200	209	323	324	34	36	10	10

Positive Reference item

Strain	TA98		TA100		TA102		TA1535		TA1537
S9-Mix	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
	2-nitro-fluorene	Benzo[a] pyrene	sodium azide	2-amino-anthracene	Mitomycin C	Benzo[a] pyrene	sodium azide	2-amino-anthracene	9-amino-acridine	Benzo[a] pyrene
Mean	158.8	157.6	954.0	949.5	1020.7	1015.0	144.5	144.5	75.0	75.9
SD	26.2	26.9	102.1	104.3	90.8	95.1	44.7	47.4	26.9	26.4
Min	102	104	701	703	756	759	62	67	28	31
Max	293	291	1365	1238	1263	1311	406	404	185	184

Tables 3 -6: Summarized data from the plate incorporation and preincubation tests (with and without metabolic activation)

Test item (µg/plate)		Plate incorporation test without metabolic activation
		Number of revertant colonies
		TA-98	TA-100	TA-102	TA-1535	TA-1537
		Mean values±SD
TA-1
31.6	mean	25.3	160.0	255.3	17.7	8.0
	SD	2.5	1.0	4.6	1.2	1.7
100	mean	28.0	149.3	252.7	16.7	6.0
	SD	0.0	12.2	3.1	2.3	1.0
316	mean	30.3	166.3	269.3	24.0	6.7
	SD	7.1	13.7	9.9	4.4	1.5
1000	mean	27.3	162.7	287.0	16.3	9.3
	SD	2.1	9.0	8.2	4.0	0.6
3160	mean	26.0	171.0	264.0	19.7	4.7
	SD	1.0	7.9	15.7	0.6	1.2
5000	mean	28.0	125.3	265.3	21.3	6.0
	SD	7.8	0.6	5.9	4.2	0.0
Negative reference item 100µL/plate)	mean	28.7	188.0	283.7	25.0	5.3
	SD	3.2	4.4	5.0	3.6	2.1
Positive reference item		2-Nitrofluorene	Sodium azide	Mitomycin C	Sodium azide	9-Amino-acridine
Concentration [µg/plate]		10	10	10	10	100
	mean	181.7	1105.3	1100.3	174.3	52.0
	SD	15.0	86.4	29.9	3.2	16.5

Test item (µg/plate)		Plate incorporation test with metabolic activation
		Number of revertant colonies
		TA-98	TA-100	TA-102	TA-1535	TA-1537
		Mean values±SD
TA-1
31.6	mean	28.7	172.0	270.0	17.7	6.7
	SD	0.6	14.8	4.0	2.1	0.6
100	mean	31.3	163.7	261.3	16.3	6.3
	SD	5.8	9.1	2.1	0.6	0.6
316	mean	24.3	173.3	270.7	22.7	7.0
	SD	3.2	9.1	1.2	0.6	0.0
1000	mean	34.7	167.3	271.7	24.0	4.3
	SD	2.5	15.3	10.1	3.5	2.3
3160	mean	32.3	174.3	273.0	19.7	6.3
	SD	4.6	0.6	16.5	5.1	0.6
5000	mean	27.7	185.7	272.0	17.0	3.3
	SD	0.6	2.1	9.2	5.3	0.6
Negative reference item 100µL/plate)	mean	32.7	175.3	276.7	22.7	4.7
	SD	6.0	2.5	14.4	3.2	1.2
Positive reference item		Benzo[a] pyrene	2-Amino-anthracene	Benzo[a] pyrene	2-Amino-anthracene	Benzo[a] pyrene
Concentration [µg/plate]		10	2	10	2	10
	mean	178.3	1129.7	1101.3	156.7	60.7
	SD	5.5	91.6	9.5	35.4	18.8

Test item (µg/plate)		Preincubation test without metabolic activation
		Number of revertant colonies
		TA-98	TA-100	TA-102	TA-1535	TA-1537
		Mean values±SD
TA-1
31.6	mean	37.7	185.3	283.3	20.3	5.7
	SD	1.5	4.9	0.6	2.1	2.3
100	mean	26.0	184.7	290.0	17.7	7.7
	SD	0.0	5.5	7.5	7.4	2.5
316	mean	34.3	177.7	277.3	22.0	4.7
	SD	6.4	4.2	28.1	3.6	1.2
1000	mean	38.3	171.3	271.0	19.0	4.3
	SD	3.5	0.6	1.7	2.6	0.6
3160	mean	27.3	178.0	269.7	23.0	4.3
	SD	2.5	12.1	22.1	3.6	1.5
5000	mean	30.7	178.3	249.7	26.0	6.0
	SD	4.0	21.4	5.7	1.0	1.0
Negative reference item 100µL/plate)	mean	34.3	189.0	284.3	22.0	6.0
5.1	SD	6.8	3.6	5.1	1.0	1.0
Positive reference item		2-Nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-Amino-acridine
Concentration [µg/plate]		10	10	10	10	100
	mean	150.0	932.7	1019.7	119.0	32.3
	SD	13.9	88.5	40.3	7.5	0.6

Test item (µg/plate)		Preincubation test without metabolic activation
		Number of revertant colonies
		TA-98	TA-100	TA-102	TA-1535	TA-1537
		Mean values±SD
TA-1
31.6	mean	30.0	182.7	283.0	15.3	5.7
	SD	3.5	9.3	7.9	2.3	0.6
100	mean	35.0	165.3	275.7	14.7	5.7
	SD	5.2	23.7	24.5	0.6	0.6
316	mean	33.3	169.3	277.7	14.3	5.3
	SD	6.7	0.6	7.8	1.5	1.2
1000	mean	31.7	165.3	269.3	18.3	3.7
	SD	1.2	12.4	3.1	2.5	0.6
3160	mean	34.0	148.7	259.0	14.3	5.0
	SD	1.0	17.6	6.6	2.3	1.0
5000	mean	35.7	149.7	280.0	15.0	4.0
	SD	1.5	26.9	10.0	1.0	1.7
Negative reference item 100µL/plate)	mean	39.0	189.0	287.3	15.0	5.7
	SD	2.6	15.6	9.0	4.4	1.2
Positive reference item		2-Nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-Amino-acridine
Concentration [µg/plate]		10	10	10	10	100
	mean	135.7	944.0	980.7	121.7	61.7
	SD	20.3	100.5	60.5	3.5	22.3

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, the test item tested up to the top concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.