N,N,N'-Trimethylbis(2-aminoethyl)ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb 2019 - 08 Mar 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)

Version / remarks:

1997-07-21

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
- Name of test substance: Ethanamine, N,N-dimethyl-2-[2-(methylamino)ethoxy]-
- Test substance No.:19/0034-1
- Lot/batch No.of test material: 08324216K0
- Expiration date of the lot/batch: 01 Nov 2020
- Purity: 86.6 corr. area-% (GC, DB-5), 86.7 corr. area-% (GC, RTX-35) water: 0.07 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (under N2 conditions)
- Stability under test conditions: The stability of the test substance under storage conditions
is guaranteed until 01 Nov 2020 as indicated by the sponsor, and the sponsor holds this responsibility

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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 water (= ultrapure water).
To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted from the stock solution 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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2900 and 5800 µg/plate (SPT and PIT)
In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate were generally selected as maximum
test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble
test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be
tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.8 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

Due to the good solubility of the test substance in water, water was used as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other:

Details on test system and experimental conditions:

Details on test system and experimental conditions
STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) was based on the
method of Ames et al.
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 or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxic
ology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were
counted.
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 d
etermination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the r
emaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicol
ogy, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were
counted.

PREINCUBATION TEST:
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima
et al.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic
activation) or phosphate buffer (without 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 for 48 – 72 hours in the dark, the bacterial colonies were counted.

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
The number of revertant colonies in the negative controls was within the range of the historical
negative control data for each tester strain
The sterility controls revealed no indication of bacterial contamination
The positive control substances both with and without S9 mix induced 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 were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubli
ng (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 was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control
data under all experimental conditions in at least two experiments carried out independently of each
other.

Statistics:

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

Toxicity
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed only using tester strain TA 1535 with and without S9 mix in the standard plate test at a concentration of 5800 µg/plate.
In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ revertants) was observed depending on the strain and test conditions at and above 333 µg/plate.

Decreased revertant numbers were observed at following concentrations (µg/plate):

Experiment S9 TA 1535 TA 100 TA 1537 TA 98 E.coli
1st-SPT Without 5800 - - - -
With 5800 - - - -
2nd-PIT Without 333 – 5800 2900 – 5800 333;
2900 - 5800 2900 – 5800 -
With - - 5800 5800 -
- = no adverse effect observed

Reduced background growth was observed at following concentrations (µg/plate):

Experiment S9 TA 1535 TA 100 TA 1537 TA 98 E.coli
1st-SPT Without -
With -
2nd-PIT Without 5800
With - - 5800 - -
- = no adverse effect observed

Conclusions:

Under the experimental conditions chosen here, it is concluded that Ethanamine, N,N- dimethyl-2-[2-(methylamino)ethoxy]- is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance Ethanamine, N,N-dimethyl-2-[2-(methylamino)ethoxy]-was tested for its mutagenic potential based on thea bility to induce pointmutations in selectedloci 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 - 5800 µg/plate (SPT) 33 µg - 5800 µg/plate (PIT)
TEST CONDITIONS:	Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).
SOLUBILITY:	No precipitation of the test substance was observed with and without S9 mix.
TOXICITY:	A bacteriotoxic effect was observed depending on the strain and test conditions at and above 333 µg/plate (for details see item4.2.).

MUTAGENICITY:

 A relevant increase in the number of his+ or trp+revertants(factor≥2:TA100,TA98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizingsystem.

CONCLUSION:

 Under the experimental conditions of this study, the test substance Ethanamine, N,N- dimethyl-2-[2-(methylamino)ethoxy]-is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

The available experimental test data concerning genetic toxicity are reliable but not sufficient for the purpose of classification under Regulation (EC) No. 1272/2008 (only an Ames test is available for assessment which was negative). Therefore, no classification is warranted concerning genetic toxicity due to lacking data.