Bis(2-ethylhexyl)amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria: OECD 471, Ames-Test (2019) - with or without metabolic activation - negative.

- Gene mutation in mammalian cells: OECD 476 (In Vitro Mammalian Cell Gene Mutation Test; HPRT locus) (BASF, 2012) - with or without metabolic activation - negative.

- Gene mutation in mammalian cells: OECD 487 (In vitro micronucleus test) (BASF, 2013) - with or without metabolic activation - the test substance is considered neither to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity)

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:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- source of S9 : Phenobarbital/b-naphthoflavone induced rat liver S9 fraction

- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1977).

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Due to strong cytotoxic effects the maximum concentration of 5000 µg/plate was reduced to 2500 µg/plate in some strains. The concentration range included two logarithmic decades.

The following concentrations were tested in pre-Experiment/ Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The following concentrations were tested in experiment II:
TA 1535: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 1537 & TA 98 : 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 100 & WP2 uvrA
(without S9 mix): 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 100 & WP2 uvrA
(with S9 mix): 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
On the day of the experiment, the test item Bis(2-ethylhexyl)amine was dissolved in Ethanol (purity > 99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981).
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Characterisation of the Salmonella typhimurium Strains and Escherichia coli Strain:
The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
The strain Escherichia coli WP2 and its derivatives carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed in Envigo CRS GmbH according to Ames et al. (1977) and Maron and Ames (1983). Thus, it is ensured that the experimental conditions set down by Ames are fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).


Precultures:
The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Nutrient Broth
5 g NaCl
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37°C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).

Plates with selective agar (without histidine/tryptophan) were used.

Overlay Agar:
The overlay agar contains per litre:
for Salmonella typhimurium: for Escherichia coli:
7.0 g Agar Agar 7.0 g Agar Agar
6.0 g NaCl 6.0 g NaCl
10.5 mg L-HistidinexHClxH2O 10.2 mg Tryptophan
12.2 mg Biotin


Pre-Experiment for Toxicity:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.


Experimental Performance
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution
(positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension,
2000 µL Overlay agar
Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
50 µL Test solution at each dose level (solvent control),
100 µL Reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension,
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL (in experiment I) or 50 µL (in experiment II) of the stock solution, 500 µL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Data Recording:
The colonies were counted using a validated computer system, which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to reduced background growth the colonies were partly counted manually.

Evaluation criteria:

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

The test item Bis(2-ethylhexyl)amine was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                
TA 1535:                                         10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 1537 & TA 98 :                        1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 100 & WP2 uvrA
(without S9 mix):                          10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 100 & WP2 uvrA
(with S9 mix):                                1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate without S9 mix and from 333 µg/plate to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	100 – 5000	100 – 5000	333 – 5000	333 – 5000
TA 1537	100 – 5000	100 – 5000	100 – 2500	100 – 2500
TA 98	100 – 5000	333 – 5000	333 – 2500	333 – 2500
TA 100	100 – 5000	333 – 5000	100 – 5000	100 – 2500
WP2uvrA	333 – 5000	333 – 5000	333 – 5000	333 – 2500

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	2500 – 5000	2500 -5000	1000 – 5000
TA 1537	1000 – 5000	1000 – 5000	333 – 2500	333 – 2500
TA 98	1000 – 5000	1000 – 5000	333 – 2500	333 – 2500
TA 100	2500 – 5000	333 – 5000	333 – 5000	333 – 2500
WP2 uvrA	2500 – 5000	1000 – 5000	1000 – 5000	1000 – 2500

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bis(2-ethylhexyl)amine at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of Bis(2-ethylhexyl)amine to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                
TA 1535:                                         10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 1537 & TA 98 :                        1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 100 & WP2 uvrA
(without S9 mix):                          10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
TA 100 & WP2 uvrA
(with S9 mix):                                1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate without S9 mix and from 333 µg/plate to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Strong toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Bis(2-ethylhexyl)amine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion,it can be stated that[RJ1] during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Bis(2-ethylhexyl)amine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 487 (in vitro MN-test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% fetal calf serum (FCS), 1% penicillin / streptomycin (10000 IU / 10000 μg/mL), 1% amphotericine B (250 μg/mL). During exposure to the test substance for 4 hours, MEM medium was used without FCS supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital / beta-naphthoflavone induced rats

Test concentrations with justification for top dose:

- 1st experiment, without S9 mix, 4 h exposure: 0.78, 1.56, 3.13, 6.25, 12.50, 25.00 μg/mL (evaluated: 1.56, 3.13, 6.25 μg/mL);
- 1st experiment, with S9 mix, 4 h exposure: 1.56, 3.13, 6.25, 12.50, 25.00, 50.00 μg/mL (evaluated: 6.25, 12.50, 25.00 μg/mL);
- 2nd experiment, without S9 mix, 24 h exposure: 0.47, 0.94, 1.88, 3.75, 5.00, 7.50 μg/mL (evaluated: 1.88, 3.75, 5.00 µg/mL);
- 2nd experiment, with S9 mix, 4 h exposure (not valid): 4.69, 9.38, 18.75, 37.50, 50.00 μg/mL (evaluated: 4.69, 9.38, 18.75 µg/mL);
- 3rd experiment, with S9 mix, 4 h exposure: 2.34, 4.69, 9.38, 18.75, 37.50, 50.00 μg/mL (9.38, 18.75, 37.50 µg/mL);

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (final concentration in culture medium was 1% (v/v))
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic assay 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:

cyclophosphamide

ethylmethanesulphonate

Remarks:

ethyl methanesulfonate (EMS): 500 and 600 μg/mL in MEM without FCS (2.5 or 3.0 mg/mL); cyclophosphamide (CPP): 2.5 μg/mL in MEM without FCS (12.5 μg/mL);

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 6 h
- Exposure duration: 4 h (experiment I, without S9 mix; experiment I, II & III, with S9 mix); 24 h (experiment II, without S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

STAIN: The slides were stained in Wrights solution (modified May-Gruenwald solution) for about 3 minutes. After being rinsed once in Titrisol solution pH 7.2, the slides were counterstained with 2.6% (v/v) Giemsa/Titrisol solution pH 7.2 for about 20 minutes. After being rinsed twice in Titrisol solution pH 7.2 and clarified in xylene, the slides were mounted using Corbit-Balsam.

NUMBER OF REPLICATIONS: at least 2 cultures (four slides)

NUMBER OF CELLS EVALUATED: at least 1000 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC), proliferation index

OTHER: Cell morphology

Evaluation criteria:

The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis. Cultures with few isolated cells were not analysed for micronuclei.

Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data.
- The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced cell counts was observed at least in the highest applied concentration in all experiments; cell attachment / morphology was adversely influenced at several test groups;

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No influence observed.
- Effects of osmolality: No influence observed.
- Water solubility: insoluble
- Precipitation: No precipitation observed.


RANGE-FINDING/SCREENING STUDIES: In the pretests for toxicity based on the purity and the molecular weight of the test substance 2500 μg/mL (approx. 10 mM) Di-(2-ethylhexyl)amine was used as top concentration. The cells were prepared at a harvest time of 24 hours (about 2 cell cycles) after 4 and 24 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix. The pretests were performed following the method described for the main experiment. As indication of test substance toxicity relative increase in cell count (RICC) and cell attachment (morphology) were determined for dose selection. In the pretests various additional parameters (pH, osmolarity, solubility) were checked or determined for all or at least some selected doses. Based on the observations and toxicity data of both pretests 50.0 μg/mL (approx. 0.2 mM) Di-(2-ethylhexyl)amine was used as top concentration in the 1st experiment of this cytogenetic study. In the pretests the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in the vehicle acetone was observed in the stock solution (Test group: 2500 μg/mL). In culture medium test substance precipitation occurred at 2500 μg/mL 4 hours after start of treatment in the absence of S9 mix and at 625 μg/mL and above in the presence of S9 mix. At 24 hours continuous treatment in the absence of
S9 mix precipitation of the test substance in culture medium occurred at 625 μg/mL and above at the end of exposure period. In the 1st pretest after 4 hours treatment with and without S9 mix and after 24 hours continuous treatment in the absence of S9 mix strong cytotoxicity occurred at all doses
applied, except at 312.5 μg/mL (highest soluble concentration). In the 2nd pretest after 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced RICC of about or below 40 - 50% was observed from 12.5 μg/mL onward. In addition, in the presence of S9 mix, clearly reduced relative increase in cell count was observed after treatment with 25.0 μg/mL and above. Besides, in the pretest with 24 hours continuous treatment in the
absence of S9 mix, the relative increase in cell count was clearly reduced after treatment with 6.25 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity indicated by reduced PI values was observed at the test groups scored for cytogenetic damage. In addition, in all main experiments in the absence and the presence of S9 mix growth inhibition indicated by reduced cell counts was observed at least in the highest applied concentration. In detail, in the absence of S9 mix the RICC was clearly reduced below 50% of control from 12.5 μg/mL onward (-9.4%) after 4 hours treatment in the 1st experiment and at 7.5 μg/mL (21.8%) after 24 hours treatment in the 2nd experiment. Additionally, in the presence of metabolic activation strongly reduced cell numbers were obtained at 50 μg/mL (-8.8% and -6.3%) in the 1st and 3rd experiment, respectively. In the invalid experimental part a strongly reduced RICC was found from 37.5 μg/mL onward (-9.3%). In this study, cell attachment / morphology was adversely influenced (grade > 2) at several test groups. In detail, in the absence of S9 mix the cell attachment was clearly reduced from 12.5 μg/mL onward after 4 hours treatment in the 1st experiment. Additionally, in the presence of metabolic activation strongly reduced cell quality was observed at 50 μg/mL in the 1st and 3rd experiment. In the invalid experimental part cell attachment / morphology was adversely influenced from 37.5 μg/mL onward. The slides of all these test groups were not scorable for cytogenetic damage. Besides, the slides at 7.5 μg/mL of the 2nd experiment without S9 mix were also not scorable due to low quality.

Remarks on result:

other: all strains/cell types tested

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index	RICC in %	Micronucleated cells* in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Acetone (1%)	2.39	100.0	0.6
		EMS (500 µg/mL)	2.52	n.t.	2.9^s
		0.78	n.d.	69.7	n.d.
		1.56	2.47	85.0	0.5
		3.13	2.54	75.4	0.5
		6.25	2.39	53.6	0.4
		12.50	n.s.	-9.4	n.s.
		25.00	n.s.	-9.8	n.s.
Exposure period 24 hrs without S9 mix
II	24 hrs	Acetone (1%)	2.63	100.0	0.7
		EMS (500 µg/mL)	2.13	n.t.	14.7^s
		0.47	n.d.	100.6	n.d.
		0.94	n.d.	92.9	n.d.
		1.88	2.51	81.7	0.5
		3.75	2.39	75.4	0.8
		5.00	2.31	59.1	1.0
		7.50	n.s.	21.8	n.s.
Exposure period 4 hrs with S9 mix
I	24 hrs	Acetone (1%)	2.34	100.0	0.8
		CPP (2.5 µg/mL)	2.04	n.t.	9.3^s
		1.56	n.d.	123.6	n.d.
		3.13	n.d.	119.2	n.d.
		6.25	2.24	127.0	1.1
		12.50	2.09	108.8	1.1
		25.00	2.21	60.8	0.9
		50.00	n.s.	-8.8	n.s.
Exposure period 4 hrs with S9 mix
II	24 hrs	Acetone (1%)	1.87	100.0	1.3
		CPP (2.5 µg/mL)	1.36	n.t.	1.5^s
		4.69	1.86	125.0	0.9
		9.38	1.99	127.6	1.7
		18.75	2.15	106.4	1.6
		37.50	n.s.	-9.3	n.s.
		50.00	n.s.	-15.4	n.s.
III	24 hrs	Acetone (1%)	2.12	100.0	0.9
		CPP (2.5 µg/mL)	1.70	n.t.	2.5^s
		2.34	n.d.	94.9	n.d.
		4.69	n.d.	120.6	n.d.
		9.38	2.14	108.0	0.8
		18.75	2.17	130.0	1.3
		37.50	2.43	56.2	1.4
		50.00	n.s.	-6.3	n.s.
^sNumber of micronucleated cells statistically significantly higher than corresponding control values
n.d. Not determined
n.s. Not scorable due to strong cytotoxicity
n.t. Not tested

In the 2nd experiment the positive control CPP showed an unexpected low aneugenic response (1.5% micronucleated cells) in the experimental part with metabolic activation, which was below the historical positive control data range (2.3 – 23.5% micronucleated cells). So, this part of the study did not fulfill the criteria for validity and, thus, it was excluded from the assessment of the genotoxic potency of the test substance. A repeat experiment, designated 3rd experiment, was performed to confirm the results of the 1st experiment in the presence of metabolic activation.

In three valid experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the micronucleus frequencies (0.4 – 1.4% micronucleated cells) were close to the concurrent vehicle control values (0.6 – 0.9% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells). Therefore, the dose dependencies observed in the 2nd experiment in the absence of S9 mix and in the 3rd experiment in the presence of S9 mix has to be regarded as biologically irrelevant.

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that Di-(2-ethylhexyl)amine has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Executive summary:

The substance Bis(2-ethylhexyl)amine was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity).

Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

On regard of the requirements of the current guidelines, and taking into account the data and observations of two pretests and the cytotoxicity actually found in the main experiments, the following doses were tested.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 0.78; 1.56; 3.13; 6.25; 12.50 and 25.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 1.56; 3.13; 6.25; 12.50; 25.00 and 50.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 0.47; 0.94; 1.88; 3.75; 5.00 and 7.50 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix (not valid)

0; 4.69; 9.38; 18.75; 37.50 and 50.00 μg/mL

3rd Experiment

4 hours exposure, 24 hours harvest time, with S9 mix

0; 2.34; 4.69; 9.38; 18.75; 37.50 and 50.00 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

The 2nd Experiment in the presence of S9 mix did not meet the criteria of validity due to lacking positive control response, thus, a 3rd Experiment with S9 mix was performed. Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or proliferation index (PI) was observed at least at the highest applied test substance concentration in all experimental parts of this study.

On the basis of the results of the present study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, Bis(2-ethylhexyl)amine is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt-locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital and beta-naphthoflavone induced rat livers

Test concentrations with justification for top dose:

- Experiment I (-S9, 4 h): 0.14, 0.28, 0.56, 1.1, 2.3, 4.5, 9.0, 18.0, 36.0, 72.0 µg/mL (concentrations evaluated: 0.56, 1.1, 2.3, 9.0, 18.0 µg/mL);
- Experiment I (+S9, 4 h): 1.1, 2.3, 4.5, 9.0, 18.0, 36.0 µg/mL (concentrations evaluated: 2.3, 4.5, 9.0, 18.0, 36.0 µg/mL);
- Experiment II (-S9, 24 h): 0.14, 0.28, 0.56, 1.1, 2.3, 4.5, 9.0, 18.0, 36.0, 72.0 µg/mL (concentrations evaluated: 0.28, 0.56, 1.1, 2.3, 4.5 µg/mL);
- Experiment II (+S9, 4 h): 0.14, 0.28, 0.56, 1.1, 2.3, 4.5, 9.0, 18.0, 36.0, 72.0 µg/mL (concentrations evaluated: 2.3, 4.5, 9.0, 18.0, 36.0 µg/mL);

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF, final concentration of THF in the culture medium was 0.5 %)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

-S9: 0.150 mg/mL EMS (dissolved in nutrient medium); +S9: 1.1 µg/mL DMBA (dissolved in DMSO);

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: 1st Experiment (- / + S9 mix): 4 h; 2nd Experiment (- S9 mix): 24 h; 2nd Experiment (+ S9 mix): 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days

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

NUMBER OF REPLICATIONS: 2 cultures per concentration

DETERMINATION OF CYTOTOXICITY:
cloning efficiency, cell density

Evaluation criteria:

Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
1.) The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
2.) The positive control substances should produce a significant increase in mutant colony frequencies.
3.) The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of results:
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 within the laboratory's historical control data range, 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. 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 relevance and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(Experiment I (-S9, 4h): at 2.3 μg/mL and above; Experiment I / Experiment II (+S9, 4 h): 36.0 μg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Adjusted to neutral with 2 N HCl; therefore, no effects observed;
- Effects of osmolality: no effects observed
- Precipitation: no precipitation observed

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this
pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). The range finding pre-experiment was performed using a concentration range of 18.9 to 2420 μg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects were observed at 605.0 μg/mL and above after 24 hours treatment in the absence of metabolic activation. No precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment. In the pre-experiment the pH at the two highest concentrations was adjusted to neutral with 2 N hydrochloric acid. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item in the pre-experiment. Based on the results of the pre-experiment the concentrations of the first experiment were selected. However, strong toxic effects occurred already at intermediate concentrations and the experiment was prematurely terminated. The concentration range of the repeat experiment and experiment II was adjusted to lower concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures were observed in the first experiment at 2.3 μg/mL and above without metabolic activation and at 36.0 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above were noted at 9.0 μg/mL and above in the absence and at 36.0 μg/mL and above in the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment I - 4 h exposure - With Metabolic Activation
Concentration	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor
[µg/mL]
	Culture I					Culture II
0 (THF)	100.0	100.0	100.0	10.9	1.0	100.0	100.0	100.0	25.6	1.0
2.3	96.0	85.6	59.3	18.9	1.7	99.2	97.2	91.3	33.1	1.3
4.5	98.1	95.1	60.9	17.4	1.6	101.4	116.3	89.8	16.6	0.7
9.0	95.4	106.9	59.7	22.8	2.1	100.7	105.2	99.0	14.1	0.6
18.0	97.9	109.4	72.3	13.5	1.2	102.4	104.1	95.6	18.1	0.7
36.0	0.0	40.5	62.9	16.8	1.5	0.0	29.5	88.9	46.2	1.8
DMBA, 1.1	87.7	77.4	61.0	731.2	67.1	86.0	100.4	55.2	1683.8	65.9
DMBA = Dimethylbenzanthracene
Table 2: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor
[µg/mL]
	Culture I					Culture II
0 (THF)	100.0	100.0	100.0	8.0	1.0	100.0	100.0	100.0	22.9	1.0
0.56	96.7	72.8	77.2	13.5	1.7	93.4	84.4	86.8	23.0	1.0
1.1	87.5	58.7	75.7	6.7	0.8	81.8	91.4	91.4	14.5	0.6
2.3	23.9	24.9	74.5	9.1	1.1	79.5	30.5	92.6	21.9	1.0
9.0	0.0	20.5	74.2	7.7	1.0	67.1	16.8	84.5	10.8	0.5
18.0	0.0	17.2	75.5	7.1	0.9	0.1	20.7	82.1	31.6	1.4
EMS, 150	104.1	46.5	73.5	86.3	10.8	92.8	53.5	85.3	164.7	7.2
EMS = Ethyl methane sulphonate
Table 3: Experiment II - 4 h Exposure - With Metabolic Activation
Concentration	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor
[µg/mL]
	Culture I					Culture II
0 (THF)	100.0	100.0	100.0	24.6	1.0	100.0	100.0	100.0	15.0	1.0
2.3	94.0	137.3	101.6	12.6	0.5	93.5	133.3	79.3	17.8	1.2
4.5	100.0	104.0	101.3	10.3	0.4	96.6	113.6	80.7	23.4	1.6
9.0	98.0	130.1	102.5	17.8	0.7	97.8	107.9	89.5	30.8	2.1
18.0	92.0	115.1	108.2	23.1	0.9	86.5	109.4	82.1	19.9	1.3
36.0	4.1	124.1	107.6	9.8	0.4	2.8	103.1	92.6	29.0	1.9
DMBA, 1.1	72.9	97.2	98.7	479.6	19.5	78.8	82.4	84.6	628.4	41.8
DMBA = Dimethylbenzanthracene
Table 4: Experiment II - 24 h exposure - Without Metabolic Activation
Concentration	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor	Relative cloning efficiency I [%]	Relative Cell Densitiy [%]	Relative cloning efficiency II [%]	Mutant colonies / 10^6 cells	Induction factor
[µg/mL]
	Culture I					Culture II
0 (THF)	100.0	100.0	100.0	20.0	1.0	100.0	100.0	100.0	35.4	1.0
0.28	94.9	85.0	102.0	15.9	0.8	91.8	81.5	155.5	18.7	0.5
0.56	103.4	82.1	105.6	27.7	1.4	94.0	94.2	167.7	26.2	0.7
1.1	97.0	89.8	120.5	19.9	1.0	100.4	86.4	176.3	26.3	0.7
2.3	99.8	93.8	107.3	27.9	1.4	94.9	105.7	96.2	22.0	0.6
4.5	90.5	80.7	120.6	16.8	0.8	87.4	81.5	198.8	23.7	0.7
EMS, 150	103.0	101.8	84.5	91.6	4.6	101.2	99.9	176.1	316.0	8.9
EMS = Ethyl methane sulphonate

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. In the second culture of experiment I the mutant frequency slightly exceeded the historical range of solvent controls (3.4 - 36.6 mutant colonies/10⁶ cells) at 36.0 μg/mL with metabolic activation (46.2 mutant colonies/10⁶ cells). However, the induction factor did not exceed the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increase was judged as biologically irrelevant.

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, Bis(2-ethylhexyl)amine is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Bis(2-ethylhexyl)amine to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

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 maximum concentration of 2420 μg/mL applied in the pre-experiment was equal to a molar concentration of about 10 mM, considering the preliminary information concerning the purity at the start of the experiment (≥ 99%). The concentration range of the main experiments was limited by cytotoxic effects. The test item was dissolved in THF.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

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, Bis(2-ethylhexyl)amine is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

GENETIC TOXICITY

1.)    In vitro data

Gene mutation in bacteria:

In a reverse gene mutation assay in bacteria (NTP, 1988), strains [TA 97, TA, 98, TA 100, TA 1535] of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 3, 10, 22, 100, 166, 333 µg/plate in the absence of mammalian metabolic activation, at concentrations of 33, 100, 222, 1000, 3333, 6666 µg/plate in the presence of Aroclor 1254-induced hamster liver S9 mix and at concentrations of 10, 33, 100, 333, 1000, 3333, 6666 µg/plate in the presence of Aroclor 1254-induced rat liver S9 mix, respectively (in all three experiments, the preincubation method was used). 

The test substance was tested up to cytotoxic concentrations (without S9 activation: 166 and 333 µg/plate; with S9 activation: 3333 and 6666 µg/plate). At non-toxic test substance concentrations, no significant increase in mutant frequency was observed, either with or without metabolic activation.The positive controls induced the appropriate responses in the corresponding strains.

A guideline conform OECD 471 (GLP) performed in 2019 revealed no signs of genetix toxicity in all species tested. High cytotoxicity was observed.

 

Gene mutation in mammalian cells:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster lung fibroblasts (V79) in vitro (BASF SE, 2012). The GLP study was conducted according to OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test; HPRT locus). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. The following doses were evaluated in this study: 1^st Experiment (-S9, 4 h): 0.56, 1.1, 2.3, 9.0, 18.0 µg/mL); 1^st Experiment (+S9, 4 h): 2.3, 4.5, 9.0, 18.0, 36.0 µg/mL; 2^nd Experiment (-S9, 24 h): 0.28, 0.56, 1.1, 2.3, 4.5 µg/mL; 2^nd Experiment / +S9, 4 h): 2.3, 4.5, 9.0, 18.0, 36.0 µg/mL). After an attachment period of 24 hours and the respective treatment period an expression phase of 7 days and a selection period of about 8 days followed. The colonies of each test group were fixed, stained with 10% methylene blue in 0.01% KOH solution and counted. Cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures were observed in the first experiment at 2.3 μg/mL and above without metabolic activation and at 36.0 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above were noted at 9.0 μg/mL and above in the absence and at 36.0 μg/mL and above in the presence of metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed up to the maximumconcentration.Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

An in vitro micronucleus test was conducted according to OECD Guideline 487 and GLP (BASF SE, 2013). The test substance was assessed in V79 cells in vitro both in the absence and the presence of a metabolizing system. The following concentrations were tested and evaluated: Experiment I (4 h / -S9): 1.56, 3.13, 6.25 μg/mL; Experiment I (4 h / +S9): 6.25, 12.50, 25.00 μg/mL; Experiment II (24 h / -S9): 1.88, 3.75, 5.00 μg/mL; Experiment II (4 h / +S9): 4.69, 9.38, 18.75 μg/mL, Experiment III (4 h / +S9): 9.38, 18.75, 37.50 μg/mL). A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within historical negative control data range for V79 cells. Both positive control substances (EMS and cyclophosphamide) led to the expected increase in the number of cells containing micronuclei. The 2nd experiment in the presence of S9 mix did not meet the criteria of validity due to lacking positive control response, thus, a 3rd Experiment with S9 mix was performed. Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or proliferation index (PI) was observed at least at the highest applied test substance concentration in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.Thus, under the experimental conditions described, the test substance is considered neither to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for selection of genetic toxicity endpoint
More than one Key study available. All are sufficient for assessment.

Short description of key information:
Genetic toxicity:
- in vitro:
Ames-Test (NTP, 1988): negative;

Ames-Test (Alkylamine Consortium, 2019): negative
HPRT test (BASF SE, 2012): negative;
In vitro MN-test (BASF SE, 2013): negative;

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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

The test substance has not to be classified under Regulation (EC) No.1272/2008.