2-ethyl-N,N-bis(2-ethylhexyl)hexylamine

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:

06/2022 - 07/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

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:

2-ethyl-N,N-bis(2-ethylhexyl)hexylamine

EC Number:

217-461-0

EC Name:

2-ethyl-N,N-bis(2-ethylhexyl)hexylamine

Cas Number:

1860-26-0

Molecular formula:

C24H51N

IUPAC Name:

tris(2-ethylhexyl)amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: B 407
- Purity: >99.8 area-% after water correction; Content of water: <0.02 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: RT
- Stability during storage: The stability of the test substance under storage conditions was guaranteed until 16 Mar 2024 as indicated by the sponsor, and the sponsor holds this responsibility.
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.
- Stability of the test material in the vehicle under test conditions: The stability of the test substance in the vehicle acetone was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in acetone. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before use.

Method

Target gene:

his, trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine auxotroph

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive (experimental conduct with records and documentation in general accordance with the GLP principles, but without GLP status).
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were washed with 150 mM KCl solution. Afterwards, the livers were weighed and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, appropriate portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- concentration S9 mix: The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4),
- quality controls of S9: To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Test concentrations with justification for top dose:

1st Experiment
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate is generally selected as maximum test dose at least in the 1st Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aceton
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98), 60 μg/plate, dissolved in DMSO (Escherichia coli WP2 uvrA), With S9 mix;

Remarks:

Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance
but without the addition of tester strain.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate (3 test plates per dose or per control)
- Number of independent experiments: 2

TREATMENT/ EXPOSURE / HARVEST:
1st Experiment: standard plate test (plate incorporation method)
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation) or 0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Reason: No mutagenicity was observed in the standard plate test.
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Toxicity defined by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was investigated for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 are not detected as toxicity in low dose groups.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations are given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.

Solubility
If precipitation of the test material is observed, it is recorded and indicated in the tables. As long as precipitation do not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Evaluation criteria:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls are within the range of the historical negative control data for each tester strain.
• The sterility controls reveal no indication of bacterial contamination. Test substance precipitation should not interfere with the scoring.
• The positive control substances both with and without S9 mix induce a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL are used.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains are within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

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 applicable

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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease in the number of his+ in the standard plate test without S9 mix at 5000 μg/plate and in the preincubation assay without S9 mix at 2500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease in the number of his+ in the standard plate test without S9 mix at 5000 μg/plate and in the preincubation assay without S9 mix at 2500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.

STUDY RESULTS
- Sterility control: The additional treated plates for sterility control showed no contamination in all performed experiments.
- Signs of toxicity: A bacteriotoxic effect (slight decrease in the number of his+) was only observed in the standard plate test testing the strains TA 1537 and TA 98 without S9 mix at 5000 μg/plate.
In the preincubation assay relevant bacteriotoxicity (slight decrease in the number of was only observed testing the strain TA1537 without S9 mix at 2500 μg/plate. Bacteriotoxicity observed in the lower concentrations (33 and 100 μg/plate) is considered as irrelevant.

HISTORICAL CONTROL DATA
- Positive historical control data: See Table 1
- Negative (solvent/vehicle) historical control data: See Table 2

Any other information on results incl. tables

Table 1: Historical Positive Controls

Strain	S9 Mix	Positive control	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	MNNG	222	82	651	7963	4223	1315.6
	With	2-AA	219	82	58	470	206	77.9
TA 100	Without	MNNG	233	82	685	5718	3211	1131.9
	With	2-AA	231	82	510	4066	1807	895.1
TA 1537	Without	AAC	234	82	283	2240	1007	321.3
	With	2-AA	231	82	45	361	149	58
TA 98	Without	NOPD	231	82	332	797	542	105.1
	With	2-AA	225	81	304	3105	1366	682
E. coli	Without	4-NQO	210	82	235	1902	852	389.5
	With	2-AA	213	82	96	317	167	39.9

 

Table 2: Historical Negative Controls

Strain	S9 Mix	Vehicle*	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	(All)	285	111	8	20	14	2.7
	With	(All)	282	111	7	29	13	3
TA 100	Without	(All)	293	111	75	150	121	12.5
	With	(All)	300	111	96	147	119	12.5
TA 1537	Without	(All)	303	111	5	14	10	1.9
	With	(All)	300	111	6	17	10	1.9
TA 98	Without	(All)	294	111	11	42	21	3.8
	With	(All)	297	111	17	35	25	3.4
E. coli	Without	(All)	273	111	18	46	32	5.7
	With	(All)	276	111	17	50	33	7.2

* water, DMSO, ethanol and acetone

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that 2-ethyl-N,N-bis(2-ethylhexyl)hexylamine is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of external metabolic activation.

Executive summary:

The test substance 2-ethyl-N,N-bis(2-ethylhexyl)hexylamine was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
DOSE RANGE: 33 μg - 5000 μg/plate (SPT), 33 μg - 5000 μg/plate (PIT)
TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without external metabolic activation (liver S9 mix from rats treated with enzyme inducers).
SOLUBILITY: Precipitation of the test substance was observed at and above 2500 μg/plate with and without S9 mix.
TOXICITY: A bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate.
MUTAGENICITY: A relevant increase in the number of or revertants (increased by a factor of 2 or above compared to the concurrent control for Salmonella typhimurium TA 100, TA 98 and Escherichia coli WP2 uvrA, or a factor of 3 or above for Salmonella typhimurium TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test with or without the addition of an external metabolizing system (liver S9 mix of rats treated with enzyme inducers).