Bis(2-ethylhexyl)amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl)amine

EC Number:

203-372-4

EC Name:

Bis(2-ethylhexyl)amine

Cas Number:

106-20-7

Molecular formula:

C16H35N

IUPAC Name:

bis(2-ethylhexyl)amine

Details on test material:

- Name of test material (as cited in study report): bis(2-ethylhexyl)amine

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.