N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

Genetic toxicity: in vitro

Administrative data

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

Study period:

From Aug. 20, 2012 to Febr.20, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The source and the target substances are aliphatic tertiary amines. Structurally, CAS 1042950-30-0 differs only in the presence of an additional (dimethylamino)propyl group linked to the central nitrogen from the source substance.
Tertiary amines can be generally represented by R-N(R´´)-R´; with elemental composition of only carbon, hydrogen and nitrogen, and where R is aliphatic hydrocarbon constituent of cyclic group.
All the amines, including tertiary, can form hydrogen bonds with water. Although tertiary amines lack a hydrogen atom attached to the nitrogen, and therefore can't form hydrogen bonds with themselves, however they can form hydrogen bonds with water molecules using the ion pair on the nitrogen. Solubility generally decreases as alkyl chains increase in length.
The source and target substances listed in Tables 1 and 2 of the read-across justification attached on this RSS, are registered as mono-constituent substances. The impurity profiles are well defined, and purity is generally high. Small changes in the impurity profile are unlikely to have significant influence on toxicological properties, which are dominated by irritation and corrosion - a feature of the target and source substances.
Thus, the chemical identity of the of the target and source substances is sufficiently clear for a meaningful comparison and assessment. 3.2 Common precursors and breakdown products
The considered substances (source and target substances) are aliphatic tertiary amines derived from ammonia [NH3] by substituting all the hydrogen atoms with alkyl groups (carbon and hydrogen containing molecular chains). Tertiary amines undergo metabolism via P-450 and MFOA, followed from C-oxidation and N-oxidation. For details see the attached documentation,

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

Only minor deviation from study plan: The additional parameters were evaluated using the existing data: suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG). No influence on the study results.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-dimethylaminoethyl)(methyl)amine

EC Number:

221-201-1

EC Name:

Bis(2-dimethylaminoethyl)(methyl)amine

Cas Number:

3030-47-5

Molecular formula:

C9H23N3

IUPAC Name:

N-[2-(dimethylamino)ethyl]-N,N',N'-trimethylethane-1,2-diamine

Details on test material:

- Name of test material (as cited in study report): Pentamethyldiethylentriamine
- Substance type: Nonaromatic amine
- Physical state: Liquid
- Analytical purity: 98.4% (w/w)
- Impurities (identity and concentrations): water 0.17% (w/w)
- Batch No.: 60/11
- Expiration date of the batch: 11/2012
- Stability under test conditions: stable
- Storage condition of test material: Test material was stored in ventilable room in original packaging.

Method

Target gene:

Thymidin kinase gene (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cells used during the experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% by volume), hereinafter referred to as growth medium.
Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone.
Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10-5 M thymidine, 1.2 x 10-4 M hypoxanthine, 3.3 x 10-5 M glycine and 7.2 x 10-7 M methotrexate.
Recovery medium is similar to cleansing medium, except that the methotrexate component is removed.
Selection medium is growth medium that contains 3 µg/mL of TFT (5-trifluorothymidine)

- 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:

other: The indicator cell was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay was isolated by American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (prepared from the livers of rats treated with Aroclor 1254) with cofactors

Test concentrations with justification for top dose:

Test concentrations for cytotoxicity testing:
39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL

Test concentrations for mutagenicity testing in the experiment without metabolic activation (3-hour or 24-hour exposure):
19.53, 39.06, 78.13, 156.3 and 312.5 µg/mL medium

Test concentrations for mutagenicity testing in the experiment with metabolic activation (3-hour exposure):
39.06, 78.13, 156.3, 312.5 and 625 µg/mL medium

Vehicle / solvent:

DMSO (dimethylsulfoxide)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: no
- Exposure duration:
3-hour or 24-hour exposure (without metabolic activation)
3-hour exposure (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: 2
without metabolic activation: 3h exposure and 24h exposure (2 experiments)
with metabolic activation: 3h exposure (2 experiments)

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

Evaluation criteria
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test item as a mutagen.

Assay acceptance criteria
An assay is considered acceptable for evaluation of the test results only if following criteria are satisfied.
a/ The mutant frequency in the negative control falls within the normal range (historical mean value).
b/ The plating efficiency of the negative control is ≥ 50%.
c/ At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without metabolic activation system..

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure), and at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary experiment
In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.
Hence, in the main study following concentration ranges were used:
a/ concentration range of 19.53 to 312.5 µg/mL (without metabolic activation, 3-hour or 24-hour exposure),
b/ concentration range of 39.06 to 625 µg/mL (with metabolic activation, 3-hour exposure).

Main study
1/Cytotoxicity:
Cytotoxicity (decreased survival) was noted
at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure),
at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure),

2/ Positive controls:
a/ Methylmethanesulfonate (at 10 or 15 µL/mL) – without metabolic activation
It caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells.
b/ 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) – with metabolic activation
It caused pronounced increases in the mutation frequency ranging from 898.28 to 3466.39 per 1E+06 clonable cells.

3/ Negative controls
a/ without metabolic activation
The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells.
b/ with metabolic activation
The values of mutation frequencies of the negative controls ranged from 89.03 to 101.24 per 1E+06 clonable cells.

4/ Test sample – Pentamethyldiethylentriamine
a/ without metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure).
b/ with metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay).

These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Ratio of small to large mutant colonies
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for test substance treated cells and from 0.42 to 1.36 for the negative controls.

Remarks on result:

other: strain/cell type: TK +/-

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Executive summary:

The test substance, Pentamethyldiethylentriamine, was tested for in-vitro mutagenicity in the Mouse Lymphoma Assay according to (EC) No. 440/2008 B.17 and OECD Guideline No.476: In Vitro Mammalian Cell Gene Mutation Test.

 

The substance was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

The vehicle DMSO (dimethylsulfoxide) served as the negative control.

 

In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.

 

Hence, in the main study the concentration range of 19.53 to 312.5 µg/mL was used in the experiments without metabolic activation with a 3-hour or 24-hour exposure time and a concentration-range of 39.06 to 625 µg/mL was used in the experiments with metabolic activation (3-hour exposure).

 

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

 

In the main study, cytotoxicity (decreased survival) was noted in the absence of metabolic activation (3-hour and 24-hor exposure) at the top concentration of 312.5 µg/mL and in the experiments with metabolic activation (3-hour exposure) at the top concentration of 625 µg/mL.

 

The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells in the experiments without metabolic activation and from 89.03 to 101.24 per 1E+06 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

 

The mutation frequencies of the cultures treated with Pentamethyldiethylentriamine ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

 

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for Pentamethyldiethylentriamine treated cells and from 0.42 to 1.36 for the negative controls.

 

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells in the case of MMS and ranging from 898.28 to 3466.39 per 1E+06 clonable cells in the case of 3-MC.

 

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.71 to 2.58 in the case of MMS.

 

Results

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.

According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.