1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

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:

April 24, 2018- May 04, 2018

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

All Salmonella typhimurium strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium. The Escherichia coli strain carries a defect in one of the genes for tryptophan biosynthesis, imposing a requirement for tryptophan in the growth medium.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

5; 15.8; 50; 158; 500; 1580; 5000 µg/plate (with and without S9 mix)
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle for test item: DMSO showed best performance and was thus used for this experiment at a maximum concentration of 17.8 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without metabolic activation, 2 µg/plate, TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation, 2 µg/plate, E.coli WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, 2 µg/plate, TA100, TA 1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation, 50 µg/plate, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation, all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The incubation of plates was performed at 36 - 38 °C for 2 days.

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met

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:

not examined

Positive controls validity:

valid

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:

not examined

Positive controls validity:

valid

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:

not examined

Positive controls validity:

valid

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate.

Ames test:
- Signs of toxicity : No toxicity to the bacteria was observed.
- Mean number of revertant colonies per plate and standard deviation : see Tables 1 and 2

Table 1: Summary 1^st Series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA1535	TA 1537	WP2 uvrA
Without Activation	DMSO		23±7	121±8	17±6	12±4	37±6
	Test item	5.00	21± 7	126 ±6	16 ±5	9±5	33 ±7
		15.8	15 ± 2	128 ±9	21 ±8	13±4	43 ±1
		50.0	20 ±1	125 ±11	18±2	12±2	42 ±6
		158	16 ±7	112 ±23	16±4	13±4	43 ±7
		500	20 ±2	121 ±5	19 ±3	9±7	38 ±3
		1580	21±7	126 ±15	23±3	10±2	39 ±9
		5000	21 ±4E	122 ±16E	16±4E	17±2E	31 ±4E
	DAUN	2.00	67 ±1
	NaN3	2.00		1478 ±27	747 ±34
	9-AA	50.0				938±130
	NQO	2.00					1857±103
With Activation	DMSO		21 ±8	122 ±13	13±4	12±4	32 ±4
	Test item	5.00	19 ±5	119±9	16±3	12±3	31 ± 11
		15.8	19 ±6	136 ±2	16±1	11±4	28 ± 12
		50.0	13±3	113±10	13±5	11±7	31±3
		158	22±10	137±5	12±2	11±3	43±14
		500	23±2	142±3	9±1	12±2	36±4
		1580	17±2	143±6	12±6	11±2	31±12
		5000	26±2E	128±6E	20±7E	9±5E	34±6E
	2-AA	2.00	195 ±38	1310±17
	2-AA	5.00			173±17	413±6
	2-AA	10.0					319±18

 Key to Positive Controls                                                        Key to Plate Postfix Codes

NaN3 Sodium azide                                                   E        Precipitation until end of experiment

2-AA 2-Aminoanthracene

9-AA 9-Aminoacridine

DAUN Daunomycin

NQO 4-Nitroquinoline-N-oxide

Table 2 Summary 2nd Series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		24±12	115±15	23±4	9±5	32±5
	Test tem	50.0	26 ±6	106 ±10	17±5	11±6	33±2
		158	35 ±9	121±7	23±1	13±7	40±5
		500	27±4	126 ±4	20 ±5	8±5	44 ±8
		1580	28±1	115 ±9	20±3	7±1	42 ±6
		5000	28 ±4E	136 ±10E	24±5E	12±5E	36 ±6E
	DAUN	2.00	243±64
	NaN3	2.00		1728±58	957±15
	9-AA	50.0				2031±574
	NQO	2.00					1785±210
With Activation	DMSO		31±11	120 ±12	15±7	10±4	40±6
	Test item	50.0	28±3	130±15	19±4	15±3	43±5
		158	25±3	132±13	18±4	10±2	43±2
		500	29±8	148±12	16±5	7±2	42±3
		1580	41±6	143±12	18±7	12±3	41±8
		5000	55±3	125±14	19±5	9±3	33±5
	2-AA	2.00	485±55	788±72
	2-AA	5.00			247±47	219±24
	2-AA	10.0					330±28

Key to Positive Controls                                                        Key to Plate Postfix Codes

NaN3 Sodium azide                                                   E        Precipitation until end of experiment

2-AA 2-Aminoanthracene

9-AA 9-Aminoacridine

DAUN Daunomycin

NQO 4-Nitroquinoline-N-oxide

 

Table 3: Historical Data

The historical data have been obtained in experiments between 01/2017 and 12/2017.

 

Negative Controls

Strain	TA 98		TA 100		TA 1537		WP2 uvrA		TA 1538
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	486	486	482	488	394	394	434	433	44	44
Number of Values	90	90	89	90	68	68	77	77	11	11
Minimum	19	20	94	90	4	6	19	28	6	11
Maximum	52	58	159	172	11	16	44	47	15	21
Mean	36	42	118	123	8	10	30	3	12	15
Standard Deviation	7.1	7.3	12.1	13.4	1.5	2.3	4.9	4.3	2.9	2.7

 

Positive Controls

Strain	TA 98		TA 100		TA 1537		WP2 uvrA		TA 1538
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3t	2-AA	9-AA	2-AA	NQO	2-AA	2-NF	2-AA
Total Plates	243	243	242	244	203	203	217	217	22	22
Number of Values	90	90	89	90	70	70	77	77	11	11
Minimum	68	112	821	437	247	72	317	154	1087	461
Maximum	779	3015	2376	3429	1485	705	2275	696	2511	1323
Mean	243	738	1550	1386	736	293	1677	353	1909	1038
Standard Deviation	134.2	508.4	213.6	724.3	284.9	161.7	381.9	129.2	470.3	285.3

 

Table 4: Optical Density

 Salmonella typhimurium

Optical Density at 650 nm (Range 0.75- 1.45)
	TA 98	TA 100	TA 1535	TA 1537
1^stseries	1.093	0.979	1.033	1.032
2^ndseries	1.123	1.006	1.075	1.037

  

Escherichia coli

Optical Density at 650 nm (Range 0.8-1.2)
	WP2 uvrA
1^stseries	0.981
2^ndseries	1.074

 

Conclusions:

The test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Executive summary:

The test item was examined for its mutagenic activity in an in vitro bacterial reverse mutation test according OECD TG 471 employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/p-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tester strains were performed using formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the requirements predetermined have been met in total and the study is considered valid. Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. No toxicity to the bacteria was observed.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was examined for its mutagenic activity in an in vitro bacterial reverse mutation test according OECD TG 471 employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/p-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.Treatments of all tester strains were performed using formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the requirements predetermined have been met in total and the study is considered valid.Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. No toxicity to the bacteria was observed.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure tothe test item in the absence and presence of S9 mix (reference 7.6.1-1).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.