4-amino-N-(2-ethylhexyl)benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the results of the presently available data, the test substance is not mutagenic in the Ames test under the experimental conditions chosen.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-04-25 to 1990-05-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study; restrictions: non-GLP , only four strains tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: All Salmonella strains (rfa-minus) mutation, UV-sensitive "uvrB-minus". For strain TA 98 and TA 100 R-factor plasmid pKM 101 (ampicillin resistance marker).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix (Aroclor 1254 treatment)

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine and 9-aminoacridine chloride monohydrate

Details on test system and experimental conditions:

I) Standard plate test with Salmonella typhimurium

METHOD OF APPLICATION:
Test tubes containing 2 mL portions of soft agar (which consists of 100 ml agar (0 .6 % agar + 0 .6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants : 0 .5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or
0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

DURATION:
- Exposure duration: After incubation at 37°C for 48 h in the dark, the bacterial colonies (his+ revertants) are counted.
- Expression time: 48 h
- Selection time: 48 h

- SELECTION AGENT: his- medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


II) Preincubation test

METHOD OF APPLICATION:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL aqua dest .
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate .
- Preincubation period: 20 min
- Exposure duration: After incubation at 37°C for 48 h in the dark, the bacterial colonies (his+ revertants) are counted.
- Expression time: 48 h
- Selection time: 48 h

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Positive controls:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535
60 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strain E. coli WP2 uvrA
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N'-nitro-N-nitroso-guanidin (ENNG) (dissolved in DMSO) for the strain E. coli WP2 uvrA

The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Occasionally a slight decrease in the number of revertant colonies was observed at doses > 2500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Incomplete solubility of test substance in DMSO from about 2500 µg/plate onward
- Precipitation: no observations reported

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table: Standard Plate Test (average results of 3 plates, respectively)

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent Control	18	19	10	12	24	37	104	104
20	17	17	11	16	27	37	107	109
100	20	17	12	17	25	37	97	122
500	19	14	10	17	27	34	107	97
2500	10	12	10	11	22	39	107	84
5000	7	11	3	8	16	39	101	98
Positive Control	1610	220	617	180	872	1430	1443	1343

 

Table: Preincubation Test (average results of 3 plates, respectively)

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent Control	22	26	10	12	24	35	86	98
20	25	25	11	10	20	32	79	88
100	26	22	11	12	20	35	89	93
500	29	31	10	13	24	35	75	92
2500	28	42	7	10	22	45	85	96
5000	26	41	6	7	15	34	91	98
Positive Control	1010	217	383	117	877	660	1078	990

 

 

 

 

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test:

The test item was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test. Tested strains were Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98 in the dose range 20 - 5000 µg/plate. Standard plate test and preincubation test both were conducted with and without metabolic activation (Aroclor induced rat liver S-9 mix). According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Justification for selection of genetic toxicity endpoint
Applicable to endpoint study, guideline-conform and conducted under GLP.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)
The available study is considered reliable and suitable for classification purposes under Directive 67/548/EEC. As a result based on this Ames test the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result based on this Ames test the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.