2-(1,3-dioxoisoindolin-2-yl)ethane-1-sulfonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria: negative with and without metabolic activation (OECD 471, GLP)

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

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

Target gene:

histidine locus

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:

preliminary study: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160, 5000 µg/plate (plate incorporation method, TA-100)
main tests: 31.6, 100, 316, 1000, 3160, 5000 µg/plate (preincubation and plate incorporation test, all strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was complete soluble in DMSO during the preliminary test

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:Two independent tests, one in agar (plate incorporation) and the other as 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

Key result

Species / strain:

S. typhimurium, other: TA-98, TA-100, TA-102, TA-1535, TA-1537

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: 1 g/L
- Precipitation: no precipitation described

RANGE-FINDING/SCREENING STUDIES: 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

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

Table 1: Summarised data: Plate incorporation test 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-3
31.6	mean	24.7	137.3	299.7	28.7	7.3
	SD	1.2	12.9	6.5	0.6	1.2
100	mean	27.7	136.3	293.7	24.7	6.7
	SD	1.5	8.4	9.1	2.1	3.5
316	mean	25.0	122.7	292.7	21.7	5.3
	SD	5.0	7.6	4.7	1.2	2.1
1000	mean	29.0	133.7	292.7	25.0	5.0
	SD	4.6	12.6	4.9	3.5	1.7
3160	mean	27.7	137.7	300.0	23.3	7.0
	SD	4.5	14.2	6.1	4.0	0.0
5000	mean	28.0	135.3	274.7	22.3	7.3
	SD	6.6	9.6	4.5	1.2	2.1
Negative reference item 100µL/plate)	mean	27.3	167.7	302.0	21.7	8.0
	SD	1.5	7.2	12.5	1.5	1.0
Positive reference item		2-Nitrofluorene	Sodium azide	Mitomycin C	Sodium azide	9-Amino-acridine
Concentration [µg/plate]		10	10	10	10	100
	mean	172.0	1047.0	1034.0	169.3	58.3
	SD	10.1	21.9	10.8	2.9	14.7

Table 2: Summarised data: Plate incorporation test with metabolic activation

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-3
31.6	mean	29.0	176.3	279.0	29.0	5.3
	SD	7.8	7.0	4.0	0.0	2.1
100	mean	28.0	165.0	294.7	20.7	8.0
	SD	7.0	7.0	6.7	0.6	1.7
316	mean	29.3	152.0	272.7	13.7	5.0
	SD	6.5	6.9	14.2	2.1	1.7
1000	mean	32.7	188.7	280.0	19.0	7.7
	SD	2.5	3.2	30.3	6.2	2.3
3160	mean	29.7	138.3	285.7	23.7	5.7
	SD	6.4	14.2	1.5	3.8	3.8
5000	mean	27.7	162.7	260.7	24.0	5.0
	SD	5.7	16.0	1.5	1.7	2.6
Negative reference item 100µL/plate)	mean	32.7	164.0	262.7	27.7	8.7
	SD	5.0	4.4	6.1	1.2	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	179.7	1076.3	1163.0	164.7	59.3
	SD	5.7	47.9	19.5	14.7	15.5

Table 3: Summarised data: Preincubation test without metabolic activation

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-3
31.6	mean	25.0	103.7	276.0	27.0	5.0
	SD	1.0	2.3	3.5	0.0	1.7
100	mean	34.0	124.0	263.7	20.3	6.0
	SD	5.6	30.3	13.5	4.0	1.7
316	mean	25.3	126.0	262.7	24.3	5.7
	SD	4.0	13.0	22.0	1.2	0.6
1000	mean	25.7	130.7	266.0	26.3	7.7
	SD	0.6	23.0	3.5	3.1	2.1
3160	mean	28.3	121.3	278.7	24.7	5.7
	SD	1.5	14.4	8.4	2.1	1.5
5000	mean	24.3	107.7	297.3	26.7	7.0
	SD	3.2	4.0	3.5	7.4	3.0
Negative reference item 100µL/plate)	mean	34.7	120.3	261.0	24.7	4.7
	SD	5.9	3.1	1.7	0.6	0.6
Positive reference item		2-Nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-Amino-acridine
Concentration [mg/plate]		10	10	10	10	100
	mean	161.7	1115.3	1151.0	175.0	68.7
	SD	1.5	14.6	18.7	7.8	13.8

Table 4: Summarised data: Preincubation test with metabolic activation

Test item (µg/plate)		Preincubation test with metabolic activation
		Number of revertant colonies
		TA-98	TA-100	TA-102	TA-1535	TA-1537
		Mean values±SD
TA-3
31.6	mean	29.0	176.3	279.0	29.0	5.3
	SD	7.8	7.0	4.0	0.0	2.1
100	mean	28.0	165.0	294.7	20.7	8.0
	SD	7.0	7.0	6.7	0.6	1.7
316	mean	29.3	152.0	272.7	13.7	5.0
	SD	6.5	6.9	14.2	2.1	1.7
1000	mean	32.7	188.7	280.0	19.0	7.7
	SD	2.5 3	3.2	30.3	6.2	2.3
3160	mean	29.7	138.3	285.7	23.7	5.7
	SD	6.4	14.2	1.5	3.8	3.8
5000	mean	27.7	162.7	260.7	24.0	5.0
	SD	5.7	16.0	1.5	1.7	2.6
Negative reference item 100µL/plate)	mean	32.7	164.0	262.7	27.7	8.7
	SD	5.0	4.4	6.1	1.2	1.2
Positive reference item		Benzo[a] pyrene	2-Amino-anthracene	Benzo[a] pyrene	2-Amino-anthracene	Benzo[a] pyrene
Concentration [mg/plate]		10	2	10	2	10
	mean	179.7	1076.3	1163.0	164.7	59.3
	SD	5.7	47.9	19.5	14.7	15.5

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver) in accordance with OECD Guideline 471 and GLP. The first experiment was carried out as a plate incorporation test and the second as a preincubation test. No  increase  in  revertant  colony  numbers  as  compared  with  control  counts  was  observed  for  the test  item,  tested  up  to  the  top  concentration  of  5000  μg/plate,  in  any  of  the  5  test  strains  in  two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test). Therefore, the test item was considered to be not mutagenic in bacteria.

Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria for germ cell mutagenicity in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).