N-ethylpropylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, Rossdorf, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-ethylpropylamine

EC Number:

243-573-4

EC Name:

N-ethylpropylamine

Cas Number:

20193-20-8

Molecular formula:

C5H13N

IUPAC Name:

N-ethylpropan-1-amine

Details on test material:

- Name of test material (as cited in study report): N-ethylpropylamine
- Physical state: Liquid, colorless, clear
- Analytical purity: 89.3%
- BASF Test Item No.: 05/0042-3
- Lot/batch No.: B987 19.05.2011
- Expiration date of the lot/batch: May 19, 2013
- Storage condition of test material: At room temperature, light protected

Method

Target gene:

Not applicable

Species / strain

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) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 Ng/mL).
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

- Experiment I (4 h; -S9 /+S9): 2.1, 4.3, 8.6, 17.2, 34.4, 68.8, 137.5, 275.0, 550.0, 1100.0 µg/mL (evaluated: 275.0, 550.0, 1100.0 µg/mL)
- Experiment IIA (24 h; -S9): 2.1, 4.3, 8.6, 17.2, 34.4, 68.8, 137.5, 275.0, 550.0, 1100.0 µg/mL (evaluated: 68.8, 137.5, 275.0 µg/mL)
- Experiment IIA (4 h; +S9): 34.4, 68.8, 137.5, 275.0, 550.0, 1100.0 µg/mL (evaluated: 68.8, 137.5, 275.0, 1100.0 µg/mL)
- Experiment IIB (24 h; -S9): 50.0, 100.0, 150.0, 200.0, 225.0, 250.0, 275.0, 300.0, 325.0, 350.0, 400.0, 550.0 µg/mL (evaluated: 250.0, 275.0, 300.0, 325.0, 350.0, 400.0, 550.0 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Remarks:

-S9: MMC: 0.3 µg/mL (in deionised water), Griseofulvin: 8.0 µg/mL (in DMSO); +S9: CPA: 15.0 µg/mL (in saline);

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I (-/+S9): 4 h, Experiment IIA (-S9): 24 h, Experiment IIA (+S9): 4 h, Experiment IIB (-S9): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two parallel cultures

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index

Evaluation criteria:

Acceptability of the assay:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronucleated cells carrying one or more micronuclei found in the solvent controls falls within the range of the historical laboratory control data range.
b) The micronucleus frequency in the positive controls is statistically significantly increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

Evaluation of results
A test item can be classified as mutagenic if: the number of micronucleated cells is not in the range of the historical control data and either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
A test item can be classified as non-mutagenic if: the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and no statistically significant or concentration-related increase in the number of micronucleated cells is observed in comparison to the respective solvent control.

Statistics:

Statistical significance was confirmed by means of the Chi square test.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment I: no mutagenicity; Experiment IIA: statistically significant increase (2.58%) at 275.0 µg/mL (dose-dependent increase); Experiment IIB: statistically significant increases at 325.0, 400.0 and 550.0 µg/mL (1.20, 2.20, 3.55 and 3.50 %)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment IIA (concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage)

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: The pH was adjusted to physiological values using small amounts of 2 N HCl in Experiment I at 1100.0 µg/mL, in Experiment IIA at 550.0 and 1100.0 µg/mL and in Experiment IIB at 550.0 µg/mL.
- Effects of osmolality: No effects observed.
- Water solubility: soluble
- Precipitation: No precipitation observed.

RANGE-FINDING/SCREENING STUDIES: With respect to the molecular weight of the test item and the preliminary purity of 85 %, 1100.0 µg/mL of N-ethylpropylamine was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 2.1 and 1100.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. No precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Since no cytotoxicity was observed up to the highest applied concentration, 1100.0 µg/mL was chosen as top treatment concentration for Experiment IIA. To verify the positive results of Experiment IIA a confirmatory experiment (Exp. IIB) was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I in the absence and presence of S9 mix, in Experiment IIA in the presence of S9 mix and in the confirmatory experiment IIB in the absence of S9 mix, no cytotoxic effects were observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

Any other information on results incl. tables

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index	Micronucleated cells* in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Water (10%)	2.97	0.95
		Mitomycin C (0.3 µg/mL)	2.52	15.40^s
		275.0	2.86	0.80
		550.0	2.78	1.50
		1100.0	2.82	0.40
Exposure period 24 hrs without S9 mix
IIA	24 hrs	Water (10%)	3.27	0.6
		Griseofulvin (8.0 µg/mL)	2.70	17.55^s
		68.8	3.20	0.50
		137.5	3.16	1.10
		275.0**	2.93	2.58^s
IIB	24 hrs	Water (10%)	2.85	0.55
		Griseofulvin (8.0 µg/mL)	2.59	8.75s
		250.0	2.89	1.10
		275.0	2.83	0.80
		300.0	2.83	0.90
		325.0	3.00	1.20^s
		350.0	2.95	2.20^s
		400.0	2.77	3.55^s
		550.0	2.42	3.50^s
Exposure period 4 hrs with S9 mix
I	24 hrs	Water (10%)	1.81	1.15
		CPA (15.0 µg/mL)	1.53	12.85^s
		275.0	1.88	1.55
		550.0	1.89	1.25
		1100.0	1.82	1.55
IIA	24 hrs	Water (10%)	2.43	1.30
		CPA (15.0 µg/mL)	1.88	8.55^s
		68.8	2.65	1.60
		137.5**	2.32	1.98
		275.0	2.15	1.65
		1100.0	2.22	1.05
* The number of micronucleated cells was determined in a sample of 2000 cells
** The number of micronucleated cells was determined in a sample of 4000 cells
^sNumber of micronucleated cells statistically significantly higher than corresponding control values

In Experiment I no mutagenicity was observed with and without S9 mix. The rates of micronucleated cells after treatment with the test item (0.40 - 1.55%) were close to the rates of the solvent control values (0.95 - 1.15%) and within the range of the laboratory historical solvent control data. In Experiment IIA in the absence of S9 mix one statistically significant increase in the percentage of micronucleated cells (2.58%), clearly exceeding the historical solvent control data range (0.05 – 1.50%) was observed after 24 hrs treatment with 275.0 µg/mL. In addition, a dose-dependent increase was observed. In Experiment IIA in the presence of S9 mix one single increase in the percentage of micronucleated cells (1.98%) was observed at 137.5 µg/mL, slightly exceeding the historical solvent control data range (0.05 – 1.70%). However, this increase was neither statistically significant nor dose-dependent and was thus regarded as biologically irrelevant. In the confirmatory experiment IIB in the absence of S9 mix statistically significant increases in micronucleated cells were observed after treatment with 325.0, 350.0, 400.0 and 550.0 µg/mL (1.20, 2.20, 3.55 and 3.50%). The three highest evaluated concentrations clearly exceeded the laboratory historical control data range of 0.05 – 1.50 %. Therefore, the positive finding could be confirmed.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item N-ethylpropylamine induced micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence of metabolic activation. Therefore, N-ethylpropylamine is considered to be mutagenic in this in vitro test system when tested up to the highest required or evaluable concentrations.