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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro studies:
- Ames assay: negative (OECD TG 471, with and without metabolic activation)
- HPRT assay: negative (OECD TG 476; V79 cells of the Chinese hamster, with and without metabolic activation)
- Chromosome aberration assay: negative (OECD TG 473; V79 cells of the Chinese hamster, with and without metabolic activation)

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

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

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

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.

Target gene:

his, trp

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 / 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

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.

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

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

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

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

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

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

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).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental: 24 Sep 2009 - 10 Dec 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

Principles of method if other than guideline:

The assay was performed in two independent experiments, using two parallel cultures each.
The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and batch 407
- Production date: 16 July 2009
- Expiration date of the batch: 16 July 2011
- Purity test date: 17-18 August 2010

- Analytical purity: 99.7%
- Lot/batch No.: Behälter 407 v. 16.07.2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility the vehicle: solubility in the vehicle determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: vortexing for formulation used

FORM AS APPLIED IN THE TEST: solution of different substance concentrations within the vehicle

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany
- Suitability of cells: V79 cell line is a trusted model in this assay (high proliferation rate, good cloning efficiancy)

For cell lines:
- Absence of Mycoplasma contamination: approved by screening of each batch
- Methods for maintenance in cell culture: subcultured twice weekly at 80 Cm2 plastic flasks with 15 mL media
- Cell cycle doubling time: 12-16 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, approved by screening of each batch
- Periodically ‘cleansed’ of spontaneous mutants: yes , approved by screening of each batch (and depressed before freezing by treatment of cells with HAT-medium)

MEDIA USED
- Type and composition of media: minimal essential medium (SEROMED, 12247 Berlin, Germany), supplemented with 10% FCS and 1% neomycin
- CO2 concentration: 1.5%
- temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced male Wistar HsdCpb:WU rat liver S9

Test concentrations with justification for top dose:

- Experiment 1: with and without S-9 mix: 112.5, 225, 450, 900, and 3600 µg/mL
- Experiment 2: with and without S-9 mix: 225, 450, 900, 1800, and 3600 µg/mL

Vehicle / solvent:

- Vehicle used: THF (tetrahydrofurane)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of THF in the culture medium was 0.5 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF APPLICATION: in suspension

TREATMENT AND HARVEST SCHEDULE:
- Seeding: experiment I: three days old exp. growing stock cultures, experiment II: five days old exp. growing stock cultures

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h (1st experiment with and without S-9 mix, 2nd experiment with S-9 mix) and 24 h (2nd experiment without S-9 mix), respectively
- Expression time (cells in growth medium): 7 days
- Selection time (incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: 10 % methylene blue in 0.01 % KOH solution (for cloning efficiency)

NUMBER OF REPLICATIONS: 5 flasks/concentration, 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency I and II
- Definition:
cloning efficiency I: survival; cloning efficiency determined immediately after treatment to measure toxicity.
cloning efficiency II: viability; cloning efficiency determined after the expression period to measure viability of the cells without selective agent.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per E+06 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

The maximal concentration of the test item equals a molar concentration of 10 mM. No relevant cytotoxic effects occurred up to the maximal concentration of 3600 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Phase separation of the test item was observed as an oily film covering the surface of the medium at 900, 1800, and 3600 µg/mL in the first experiment with and without metabolic activation.

Table 1: Summary of the results from the two hprt experiments

			relative	relative	mutant		relative	relative	mutant
	conc. µg	S9	cloning	cloning	colonies/	induction	cloning	cloning	colonies/	induction
	per m L	mix	efficiency I	efficiency II	10^6cells	factor	efficiency I	efficiency II	10^6cells	factor
			%	%			%	%
Colum n	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment			culture I				culture II
Solvent control with THF		-	100.0	100.0	14.7	1.0	100.0	100.0	12.3	1.0
Positive control with EMS	150.0	-	91.5	70.9	147.4	10.0	96.7	98.2	101.8	8.3
Test item	112.5	-	64.1	77.4	16.9	1.2	73.4	87.0	22.1	1.8
Test item	225.0	-	85.0	77.4	23.0	1.6	68.5	70.0	29.2	2.4
Test item	450.0	-	80.1	81.8	19.5	1.3	74.0	104.9	18.4	1.5
Test item	900 (p)	-	81.9	73.6	9.0	0.6	91.7	92.5	14.3	1.2
Test item	1800 (p)	-	76.8	culture was not continued^#			83.2	culture was not continued^#
Test item	3600 (p)	-	93.4	73.3	11.4	0.8	83.9	104.6	14.4	1.2
Solvent control with THF		+	100.0	100.0	18.1	1.0	100.0	100.0	5.5	1.0
Positive control with DMBA	1.1	+	24.4	71.5	1015.1	56.1	21.8	53.6	973.2	176.0
Test item	112.5	+	83.2	105.8	9.9	0.5	102.7	99.6	17.1	3.1
Test item	225.0	+	85.3	107.4	11.5	0.6	97.7	99.7	14.1	2.5
Test item	450.0	+	86.3	112.3	12.3	0.7	104.0	90.7	12.3	2.2
Test item	900 (p)	+	87.2	107.3	13.2	0.7	105.2	99.0	16.1	2.9
Test item	1800 (p)	+	86.4	culture was not continued^#			92.4	culture was not continued^#
Test item	3600 (p)	+	90.7	94.5	14.3	0.8	94.3	106.5	10.9	2.0
Experiment II / 24 h treatment			culture I				culture II
Solvent control with THF		-	100.0	100.0	4.8	1.0	100.0	100.0	22.3	1.0
Positive control with EMS	75.0	-	66.8	82.4	132.0	27.7	101.9	102.1	188.8	8.4
Test item	112.5	-	99.7	culture was not continued^##			105.6	culture was not continued^##
Test item	225.0	-	97.8	98.3	12.3	2.6	98.3	112.6	18.7	0.8
Test item	450.0	-	97.0	99.5	15.2	3.2	88.4	109.8	14.2	0.6
Test item	900.0	-	99.0	96.1	18.4	3.9	88.8	126.6	8.3	0.4
Test item	1800.0	-	99.2	101.3	12.5	2.6	94.0	103.0	17.2	0.8
Test item	3600.0	-	95.6	99.7	4.8	1.0	98.7	126.0	14.9	0.7
Experiment II / 4 h treatment
Solvent control with THF		+	100.0	100.0	11.9	1.0	100.0	100.0	4.9	1.0
Positive control with DMBA	1.1	+	97.3	93.8	403.9	33.8	97.5	79.4	143.0	29.5
Test item	112.5	+	98.1	culture was not continued^##			97.7	culture was not continued^##
Test item	225.0	+	91.8	107.9	17.1	1.4	97.5	78.6	6.1	1.3
Test item	450.0	+	106.7	98.9	18.9	1.6	89.5	90.4	8.0	1.6
Test item	900.0	+	103.6	98.1	24.3	2.0	97.7	85.3	10.9	2.3
Test item	1800.0	+	99.7	95.0	12.9	1.1	91.1	82.5	12.1	2.5
Test item	3600.0	+	103.6	90.5	13.1	1.1	92.6	87.0	11.2	2.3

#: Culture was not continued to avoid analysis of too many insoluble concentrations.

##: Culture was not continued since a minimum of only four analysable concentrations is required.

P: Phase separation visible to the unaided eye.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test substance is considered to be non-mutagenic in the HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental: 30 Sep - 10 Dec 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and batch 407
- Production date: 16 July 2009
- Expiration date of the batch: 16 July 2011
- Purity test date: 17-18 August 2010

- Analytical purity: 99.7%
- Lot/batch No.: Behälter 407 v. 16.07.2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility the vehicle: solubility in the vehicle determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: vortexing for formulation used

FORM AS APPLIED IN THE TEST: solution of different substance concentrations within the vehicle

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany
- Suitability of cells: V79 cell line is a trusted model in this assay (high proliferation rate, good cloning efficiancy)

For cell lines:
- Absence of Mycoplasma contamination: approved by screening of each batch
- Methods for maintenance in cell culture: subcultured twice weekly at 80 Cm2 plastic flasks with 15 mL media
- Cell cycle doubling time: 14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, approved by screening of each batch
- Periodically ‘cleansed’ of spontaneous mutants: yes, approved by screening of each batch (and depressed before freezing by treatment of cells with HAT-medium)

MEDIA USED
- Type and composition of media: minimal essential medium (SEROMED, 12247 Berlin, Germany), supplemented with 10% FCS, Neomycin (5 Pg/mL) and Amphotericin B (2.5 Pg/mL)
- CO2 concentration: 1.5%
- temperature: 37°C

Cytokinesis block (if used):

Colcemid based block, (0.2 µg/mL) 15.5 hours and 25.5 hours after the start of the treatment

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced male Wistar HsdCpb:WU rat liver S9

Test concentrations with justification for top dose:

Experiment I, preparation interval 18 h, exposure period 4 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment I, preparation interval 18 h, exposure period 4 h, with S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL

Experiment II, preparation interval 18 h, exposure period 18 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment II, preparation interval 28 h, exposure period 18 h, without S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL
Experiment II, preparation interval 28 h, exposure period 18 h, with S9 mix: 0.02, 0.04, 0.08, 0.2, 0.3, 0.6, 1.3, 2.5, 5.0 µL/mL

Vehicle / solvent:

- Vehicle used: THF (tetrahydrofurane)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of THF in the culture medium was 0.5 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofurane (THF)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF APPLICATION: in Quadriperm dishes which contained microscopic slides

DURATION
- Exposure duration: 4h Experiment I with and without S9 mix; Experiment II with S9 mix), 18 and 28 h (Experiment II without S9 mix), respectively
- Recovery time: 4 h (Experiment I with and without S9 mix), 24 h (Experiment II with S9 mix)
- Preparation time (incubation with colcemid): Colcemid was added to the culture medium 15.5 and 25.5 h after the start of treatment. The cells were treated, 2.5 h later, on the slides in the chambers with hypotonic solution for 20 min at 37°C. After incubation in hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1).
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 h, respectively

STAIN (cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two parallel cultures were set up in each experimental group. Two independent experiments were performed.

NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage, except for the positive control in Exp. I without S9 mix.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphases per culture was determined.

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data range.
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data range.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range.

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

5 µL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect.
- Effects of osmolarity: no effect.
- Precipitation: no test item precipitation observed.
- Other confounding effects: Phase separation occurred without S9 mix at 0.2 µL/mL and above and in the presence of S9 mix at 0.04 µL/mL and above in Experiment I. Presumably due to a longer preparation interval of 28 h, no phase separation occurred in Experiment II.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment 5.0 µL/mL of Tris-(2-ethylhexyl)amin O 2446 was applied as top concentration for treatment of the cultures. At the selected dose no influence on pH value or osmolarity was detected. Phase separation occurred at concentrations of 0.2 µL/mL and above in the absence of S9 mix and at 0.04 µL/mL and above in the presence of S9 mix. Neither precipitation nor test item induced cytotoxicity occurred. Therefore, precipitation and cytotoxicity did not interfere with the evaluation and the experiment was designated main experiment I. However, the cultures did not fulfil the acceptance criteria for cytogenetic evaluation as the number of aberrations found in the solvent controls were not within the range of the historical control data range. Therefore, Experiment I was repeated with the same maximum test item concentration.

Table 1: Summary of results of the chromosome aberration study without metabolic activation

Exp. Preparation interval		Test item concentration in µL/mL	Cell numbers in % of control	Mitotic indices in % of control	incl. gaps*	Aberrant cells in % excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control^1	100.0	100.0	3.0	2.5	1.5
		Positive control^2#	n.t.	62.9	49.0	45.0^S	33.0
		0.08	102.9	94.9	3.5	3.5	2.5
		1.3^PS	96.1	109.9	2.0	2.0	0.0
		2.5^PS	102.2	109.3	3.5	3.0	1.5
		5.0^PS	91.3	106.5	2.5	2.0	1.0
Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control^1	100.0	100.0	1.0	0.5	0.0
		Positive control^3	n.t.	59.0	19.5	18.5^S	6.0
		1.3	109.4	102.2	1.5	1.0	0.0
		2.5	109.6	126.7	2.5	2.5	0.0
		5.0	103.1	121.6	1.5	1.0	0.0
Exposure period 28 hrs without S9 mix
II	28 hrs	Solvent control^1	100.0	100.0	2.0	2.0	0.0
		Positive control^3	n.t.	54.3	28.5	28.0^S	15.0
		1.3	88.9	104.5	1.0	0.0	0.0
		2.5	83.4	106.2	0.0	0.0	0.0
		5.0	76.6	108.9	1.0	1.0	0.0

*: Inclusive cells carrying exchanges

#: Evaluation of 50 metaphases per culture

n.t.: Not tested

S: Aberration frequency statistically significant higher than corresponding control values

PS: Phase separation occurred

1: THF 0.5 % (v/v)

2: EMS 1000.0 Pg/mL

3: EMS 500.0 Pg/mL

Table 2: Summary of results of the chromosome aberration study with metabolic activation

Exp. Preparation interval		Test item concentration in µL/mL	Cell numbers in % of control	Mitotic indices in % of control	incl. gaps*	Aberrant cells in % excl. gaps*	with exchanges
Exposure period 4 hrs with S9 mix
I	18 hrs	Solvent control^1	100.0	100.0	0.5	0.5	0.5
		Positive control^2	n.t.	72.5	17.0	14.5^S	4.5
		0.02	75.0	126.9	4.0	3.5^S	1.0
		1.3^PS	90.5	101.8	2.5	2.5	0.5
		2.5^PS	68.7	106.0	2.5	2.0	0.5
		5.0^PS	94.7	119.3	2.5	2.0	0.5
II	28 hrs	Solvent control^1	100.0	100.0	1.0	0.5	0.5
		Positive control^2	n.t.	103.8	10.0	8.0^S	1.0
		1.3	96.4	106.1	0.5	0.5	0.5
		2.5	98.3	99.7	3.0	3.0^S	1.0
		5.0	93.8	97.7	1.0	0.5	0.0

*: Inclusive cells carrying exchanges

n.t.: Not tested

S: Aberration frequency statistically significant higher than corresponding control values

PS: Phase separation occurred

1: THF 0.5 % (v/v)

2: CPA 1.4 Pg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to the highest by the OECD guideline required concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in bacteria:

The test substance was tested for mutagenicity in the Ames assay according to OECD TG 471 (adopted 1981) and following the testing protocol published by Ames (1973, 1975). Different concentrations of the substance (dose range: 33 μg - 5000 μg/plate (SPT), 33 μg - 5000 μg/plate (PIT)) were tested in Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and in the Escherichia coli strain WP2 uvrA in the standard plate test (SPT) and preincubation test (PIT) both with and without external metabolic activation (liver S9 mix from rats treated with enzyme inducers). Precipitation of the test substance was observed at and above 2500 μg/plate with and without S9 mix. A bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate. A relevant increase in the number of or revertants 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). According to the results of the present study, the test substance Tri-2-ethylhexylamine is not mutagenic in the Ames test under the experimental conditions.

 

Genetic toxicity in mammalian cells:

The potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated in a study according to OECD TG 476 and GLP regulations.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration (3600 µg/mL) was equal to a molar concentration of approximately 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

In addition, the test substance, diluted in tetrahydrofuran (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The assays were conducted in accordance with OECD TG 473 and GLP regulations.

In the first experiment exposure periods of 4 hours with and without metabolic activation were employed. The preparation interval was 18 hours. In the second experiment exposure periods were 18 and 28 hours without metabolic activation, and 4 hours with metabolic activation, respectively. Accordingly, the preparation intervals were 18 and 28 hours without, and 28 hours with metabolic activation. Metabolic activation was provided by adding S-9 mix from Phenobarbital /ß-naphthoflavone induced male Wistar HsdCpb:WU rat livers.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated. The highest applied concentration (5µL/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data.

 

In this study in the absence and presence of S9 mix neither test item precipitation nor cytotoxicity was observed up to the highest applied concentration. Phase separation occurred in Experiment I in the presence and absence of metabolic activation.

In both independent experiments no clastogenicity was observed at the concentrations evaluated for cytogenetic damage either with or without metabolic activation. However, two statistically significant increases in the number of aberrant cells excluding gaps were observed after treatment with 0.02 µL/mL (3.5 % aberrant cells excluding gaps) in Experiment I in the presence of S9 mix and with 2.5 µL/mL (3.0 % aberrant cells excluding gaps) in Experiment II in the presence of S9 mix. These observations have to be regarded as biologically irrelevant, since the values were in the range of the laboratory’s historical solvent control data (Exp. I, with S9 mix: 0.0 – 4.0 % aberrant cells excluding gaps; Exp. II, with S9 mix: 0.5 – 3.0 % aberrant cells excluding gaps) and no dose-related increases were observed. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest required concentration.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480.. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.