N-ethylpropylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative (Alkylamines Consortium, 2012)

HPRT: negative (Alkylamines Consortium, 2013)

MNT in vitro: positive w/out S9, negative w/S9 (Alkylamines Consortium, 2013)

Link to relevant study records

Reference

Reference 1

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

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

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) 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

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.

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 Jul 1997)

Deviations:

yes

Remarks:

(2-aminoanthracene was used as sole indicator of the efficacy of the S9-mix)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his- or trp-gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital and β-naphthoflavone induced rats

Test concentrations with justification for top dose:

Standard plate test (SPT): 33 μg - 6000 μg/plate
Preincubation test (PIT): 10 μg - 3000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance in ultrapure water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix; 2.5 μg/plate / in DMSO (TA 1535, TA 100, TA 1537, TA 98); 60 μg/plate / in DMSO (Escherichia coli WP2 uvrA);

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S9-mix; 5 μg/plate / in DMSO (TA 1535, TA 100);

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9-mix; 10 μg/plate / in DMSO (TA 98);

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix; 100 μg/plate / in DMSO (TA 1537);

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix; 5 μg/plate / in DMSO (E. coli WP2 uvrA);

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: about 20 minutes
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer

OTHER EXAMINATIONS:
- Other: Titer determination (Titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.)

Evaluation criteria:

Acceptance criteria:

Generally, the experiment is considered valid if the following criteria are 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 within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT:>/= 3000 µg/plate; PIT: >/= 1000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT:>/= 3000 µg/plate; PIT: >/= 1000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT:>/= 3000 µg/plate; PIT: >/= 1000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT: >/= 3000 μg/plate; PIT: >/= 1000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT: >/= 3000 μg/plate; PIT: >/= 1000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions from about 3000 μg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 1000 μg/plate onward.

Table 1. Test results of experiment 1 (plate incorporation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	77 ± 7	12 ± 2	70 ± 6	23 ± 5	7 ± 1
–	33	75 ± 2	11 ± 1	71 ± 3	27 ± 7	7 ± 2
–	100	79 ± 6	12 ± 2	71 ± 7	21 ± 1	8 ± 2
–	333	79 ± 10	10 ± 2	67 ± 7	23 ± 5	7 ± 1
–	1000	87 ± 4	11 ± 1	67 ± 7	25 ± 3	6 ± 2
–	3000	38 ± 3B	8 ± 3B	42 ± 13B	12 ± 2B	4 ± 1B
–	6000	B	B	8 ± 3B	B	B
Positive controls, –S9	Name	MNNG	MNNG	4-NQO	NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	926 ± 73	1100 ± 49	1039 ± 68	738 ± 26	459 ± 16
+	0	89 ± 9	13 ± 1	73 ± 4	30 ± 5	8 ± 1
+	33	81 ± 3	14 ± 3	64 ± 3	31 ± 5	7 ± 3
+	100	88 ± 12	13 ± 2	72 ± 5	34 ± 3	8 ± 3
+	333	86 ± 15	12 ± 3	73 ± 4	27 ± 6	8 ± 2
+	1000	81 ± 8	14 ± 1	71 ± 3	28 ± 5	8 ± 3
+	3000	66 ± 5	10 ± 1	54 ± 6	10 ± 2	5 ± 2B
+	6000	6 ± 2B	B	21 ± 2B	1 ± 1B	B
Positive controls, –S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	981 ± 78	231 ± 11	276 ± 7	865 ± 31	175 ± 9

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

NOPD = 4-nitro-o-phenylenediamine

2AA = 2-aminoanthracene

AAC = 9-aminoacridine

B = reduced background growth

Table 2. Test results of experiment 2 (preincubation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	83 ± 14	11 ± 2	71 ± 6	17 ± 3	6 ± 2
–	10	78 ± 5	11 ± 2	73 ± 6	18 ± 3	6 ± 2
–	33	82 ± 8	11 ± 3	66 ± 9	20 ± 2	6 ± 2
–	100	83 ± 3	12 ± 1	70 ± 7	16 ± 2	6 ± 2
–	333	70 ± 8	10 ± 2	64 ± 4	14 ± 3	5 ± 0
–	1000	18 ± 6B	7 ± 2B	17 ± 4B	6 ± 1B	1 ± 1B
–	3000	B	B	B	B	B
Positive controls, –S9	Name	MNNG	MNNG	4-NQO	NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	878 ± 45	744 ± 26	795 ± 82	791 ± 36	459 ± 27
+	0	100 ± 9	12 ± 2	76 ± 6	25 ± 4	7 ± 2
+	10	98 ± 3	13 ± 3	74 ± 8	25 ± 3	7 ± 1
+	33	100 ± 9	12 ± 1	79 ± 6	24 ± 5	7 ± 2
+	100	98 ± 13	11 ± 2	76 ± 9	24 ± 2	6 ± 1
+	333	89 ± 3	11 ± 1	73 ± 4	20 ± 4	7 ± 1
+	1000	50 ± 10	5 ± 3	52 ± 3	12 ± 2	2 ± 2B
+	3000	14 ± 4B	3 ± 2B	18 ± 5B	3 ± 1B	B
Positive controls, –S9	Name	2AA	2AA	2-AA	2-AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	924 ± 39	130 ± 19	285 ± 13	709 ± 44	135 ± 14

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

NOPD = 4-nitro-o-phenylenediamine

2AA = 2-aminoanthracene

AAC = 9-aminoacridine

B = reduced background growth

Conclusions:

Under the experimental conditions of this study, the test substance N-ethylpropylamine is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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)

Version / remarks:

(adopted July 21, 1997)

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: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%)
- 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:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

30.6, 61.3, 122.5, 245.0, 490.0, 980.0 µg/mL (concentration evaluated: 61.3, 122.5, 245.0, 490.0, 980.0 µg/mL)

Vehicle / solvent:

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

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: EMS: 0.15 mg/mL (in medium); +S9: DMBA: 1.1 µg/ml (in DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: Experiment I: 4 h (-/+S9); Experiment II: 24 h (-S9), 4 h (+S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 19 - 20 days

SELECTION AGENT (mutation assays): 6-thioguanine (11 µg/mL)
STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cell density; cloning efficiency

Evaluation criteria:

Acceptability of the Assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. 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 is below 0.05. However, both, biological 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:

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH was adjusted to 7.2; therefore, no effects were observed
- Effects of osmolality: No effects observed.
- Water solubility: soluble
- Precipitation: Not observed.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. 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). In the range finding pre-experiment test item concentrations between 7.7 and 980 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation after 4 and 24 hours treatment. In the pre-experiment there was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item. The pH value at the maximum concentration of the pre-experiment and the main experiments was adjusted to neutral with 2 N hydrochloric acid. Based on the results of the pre-experiment, the maximum concentration of the main experiments was again 980 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% in both cultures were observed in both main experiments up to the maximum concentration of 980 µg/mL equal to approximately 10 mM with and without metabolic activation following 4 and 24 hours treatment.

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 (Water)	100.0	100.0	100.0	31.0	1.0	100.0	100.0	100.0	22.8	1.0
61.3	97.5	111.9	84.9	19.5	0.6	97.3	139.7	105.7	29.2	1.3
122.5	98.7	92.1	104.8	22.4	0.7	93.3	120.5	93.8	10.6	0.5
245.0	100.1	96.8	95.3	13.2	0.4	92.8	126.5	98.3	18.3	0.8
490.0	97.4	105.9	102.3	17.4	0.6	93.0	105.6	131.3	24.6	1.1
980.0	98.8	102.7	103.9	22.0	0.7	91.3	122.1	112.3	10.1	0.4
DMBA, 1.1	101.6	95.1	90.5	347.9	11.2	91.3	116.6	146.8	246.4	10.8
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 (Water)	100.0	100.0	100.0	19.7	1.0	100.0	100.0	100.0	10.4	1.0
61.3	104.1	78.8	89.9	24.5	1.2	96.7	76.6	137.8	10.7	1.0
122.5	100.3	55.8	101.7	22.9	1.2	95.5	58.5	123.6	7.9	0.8
245.0	98.0	68.2	89.6	20.0	1.0	99.3	64.9	120.4	6.9	0.7
490.0	102.3	63.4	93.9	14.1	0.7	97.2	61.0	111.2	11.9	1.1
980.0	97.8	45.8	100.3	13.1	0.7	92.7	89.1	114.2	13.5	1.3
EMS, 150	91.9	57.4	101.9	103.1	5.2	97.2	87.6	106.7	55.9	5.4
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 (Water)	100.0	100.0	100.0	8.5	1.0	100.0	100.0	100.0	6.1	1.0
61.3	97.7	90.2	83.9	5.1	0.6	96.7	96.7	84.2	9.1	1.5
122.5	99.6	113.7	82.3	16.4	1.9	100.4	103.4	91.0	9.1	1.5
245.0	97.7	108.3	93.6	18.5	2.2	97.1	92.4	80.7	12.4	2.0
490.0	96.8	99.4	100.3	13.1	1.5	99.1	119.5	92.8	16.0	2.6
980.0	101.1	107.4	80.0	25.4	3.0	96.5	109.4	85.1	20.9	3.4
DMBA, 1.1	97.0	83.1	91.0	369.9	43.7	94.7	86.0	78.0	302.0	49.2
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 (Water)	100.0	100.0	100.0	22.3	1.0	100.0	100.0	100.0	7.5	1.0
61.3	103.1	107.0	73.7	16.2	0.7	99.6	38.2	68.4	14.0	1.9
122.5	100.2	115.4	78.8	18.9	0.8	102.4	41.2	69.9	12.2	1.6
245.0	101.2	130.2	66.1	21.4	1.0	98.7	52.2	70.1	18.6	2.5
490.0	101.9	126.3	76.2	17.9	0.8	99.4	44.2	72.1	15.7	2.1
980.0	97.6	96.9	75.4	17.3	0.8	99.8	43.8	62.2	36.1	4.8
EMS, 150	100.4	93.6	96.1	110.6	5.0	98.2	50.3	96.4	126.3	16.8
EMS = Ethyl methane sulphonate

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of experiment II without metabolic activation at 980 µg/mL and in both cultures of experiment II with metabolic activation at 980 µg/mL. These effects however, are based upon the rather low solvent controls of 7.5, 8.5, and 6.1 mutant colonies/10⁶cells, respectively.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the second experiment with metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency remained within the historical range of solvent controls and was not reproduced in the parallel culture. Another significant trend was noted in the first experiment, culture I without metabolic activation, and in the second culture of experiment II without metabolic activation. Both of these trends however, were biologically irrelevant as they were reciprocal, going down versus increasing concentrations.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, N-ethylpropylamine is considered to be non-mutagenic in this HPRT assay.

Genetic toxicity in vivo

Description of key information

MNT in vivo: negative (Alkylamines Consortium, 2013)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April 2013 - 18 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998-08

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: mice / Crl:NMRI from Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 29.08 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M II, single housing
- Diet (e.g. ad libitum): standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland);
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to the good solubility of the test subsance in water
- Concentration of test material in vehicle: 175, 350 and 700 mg/ml, respectively for the low, mid and high dose groups
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in deionized water.
To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 (all groups) or 48 hours (for additional 5 animals in in the vehicle control and in the high dose groups)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (10 in the vehicle control and in the high dose groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide (CCP; dissolved in deionized water, 2 mg/ml) and Vincristine sulfate (VCR; dissolved in deionized water, 0.015 mg/ml)
- Route of administration: orally for CCP or intraperitoneally for VCR, each 10 ml/kg bw
- Doses / concentrations: 20 mg/kg bw for CCP and 0.15 mg/kg bw for VCR

Tissues and cell types examined:

Bone marrow; polychromatic erythrocytes, normochromatic erythrocytes,

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In a pretest for the determination of the acute oral toxicity, deaths were observed at 1 000 mg/kg body weight from one hour after test substance administration onward. At 500 mg/kg, all animals survived showing weak signs of toxicity. However, there were no distinct differences in clinical
observations between males and females. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 700 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 350 mg/kg and 175 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- The animals were anesthetized and sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissus.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS; preheated up to 37°C; about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged. The supernatant was removed and the precipitate was resuspended in fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
- Staining of the slides: the slides were stained with eosin and methylene blue, rinsing in deionized water, and then soaked in deionized water.
The slides were subsequently stained with Giemsa solution, rinsed twice in deionized water, clarifying in xylene and mounted in Corbit-Balsam.
- Microscopic evaluation: in general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of
every test group, so in total 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
Number of normochromatic erythrocytes
Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in theduration of the sacrifice interval.
Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).

OTHER:
After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.

Evaluation criteria:

Acceptance criteria: the mouse micronucleus test is considered valid if the following criteria are met:
- the quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.
Assessment criteria: a finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: (1) * p ≤ 0.05; (2) ** p ≤ 0.01. However, both biological relevance and statistical significance were considered together.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

distinct clinical signs of toxicity at both top dose groups (700 mg/kg body weight): piloerection, hunched posture, reduced general condition, respiration irregular

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Induction of micronuclei (for Micronucleus assay): according to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (175 mg/kg, 350 mg/kg and 700 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
Based on the pretest, 700 mg/kg body weight was defined as maximum tolerated dose (MTD) due to deaths observed at 1 000 mg/kg body weight. The bioavailability of the test substance in bone marrow after oral administration was clearly demonstrated by effects on the ratio of PCEs and NCEs.
In this study, after single oral administration of the vehicle deionized water the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice
intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control
animals was within the range of the historical vehicle control data for PCEs
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Table 1: Summary table – Induction of micronuclei in bone marrow cells

Test group (mg/kg bw)	Sacrifice interval (hours)	Number of animals	Micronuclei in polychromatic erothrocytes		PCEs per 2000 erythrocytes^c
			Total^a(‰)	Large^b(‰)
Vehicle control	24	5	1.2	0.0	1445
Test substance (175)	24	5	1.0	0.0	1551
Test substance (350)	24	5	0.7	0.0	1449
Test substance (700)	24	5	0.8	0.0	1226
Positive control (CCP; 20)	24	5	11.4**	0.0	1616
Positive control (VCR; 0.15)	24	5	29.0**	6.2**	1337
Vehicle control	48	5	1.2	0.1	1415
Test substance (700)	48	5	0.8	0.0	1050
^a: sum of small and large micronuclei;^b: large micronuclei (indication for spindle poison effect); ^c: calculated number of PCEs per 2000 erythrocytes (PCE + NCE) when scoring a sample of up to 10000 PCEs; **: = p ≤ 0.01

 

Conclusions:

Under the experimental conditions chosen here, the test substance N-ethylpropylamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

1) In vitro data

 

- Gene mutation in bacterial cells:

 

In a bacterial reverse mutation assay conducted according to OECD Guideline 471 and GLP, the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (REACH Alkylamines Consortium, 2012). The test concentrations ranged between 33 and 6000μg/plate in the standard plate test (triplicate; with and without S9 mix from phenobarbital and β-naphthoflavone induced rats), and between 10 and 3000μg/plate in the preincubation test (triplicate; with and without S9 mix). Sterility and vehicle controls were added, as well as positive controls. A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions from about 3000 μg/plate onward, in the preincubation assay from about 1000 μg/plate onward. No precipitation could be seen. No twice or more increase of his+ or trp+ revertants compared to the negative control value or dose-dependency was noticed. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium and E. coli mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.

 

- 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 (REACH Alkylamines Consortium, 2013). 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: 61.3, 122.5, 245.0, 490.0, 980.0 µg/mL. After an attachment period of 24 hours and the respective treatment period (Experiment I: 4 h (-/+S9); Experiment II: 24 h (-S9), 4 h (+S9)) an expression phase of 7 days and a selection period of 8 days followed. The colonies of each test group were fixed, stained with10% methylene blue in 0.01% KOH solutionand counted. No relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% in both cultures were observed in both main experiments up to the maximum concentration of 980.0 µg/mL equal to approximately 10 mM with and without metabolic activation following 4 and 24 hours treatment. 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.

 

- Micronucleus test in mammalian cells:

 

An in vitro micronucleus test was conducted according to OECD Guideline 487 and GLP (REACH Alkylamines Consortium, 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 /+S9): 275.0, 550.0, 1100.0 µg/mL; Experiment IIA (24 h; -S9): 68.8, 137.5, 275.0 µg/mL; Experiment IIA (4 h; +S9): 68.8, 137.5, 275.0, 1100.0 µg/mL, Experiment IIB (24 h; -S9): 250.0, 275.0, 300.0, 325.0, 350.0, 400.0, 550.0 µg/mL). A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. 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.In Experiment I no mutagenicity was observed with and without S9 mix. 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 value was not statistically significant. 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 and N-ethylpropylamine is considered to be mutagenic in this in vitro test system when tested up to the highest required or evaluable concentrations.

2) In vivo data:

 

An in vivo micronucleus test was conducted according to OECD Guideline 474 and GLP (REACH Alkylamines Consortium, 2013). No statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (175 mg/kg, 350 mg/kg and 700 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range. Based on the pretest, 700 mg/kg body weight was defined as maximum tolerated dose (MTD) due to deaths observed at 1 000 mg/kg body weight. The bioavailability of the test substance in bone marrow after oral administration was clearly demonstrated by effects on the ratio of PCEs and NCEs. In this study, after single oral administration of the vehicle deionized water the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data for PCEs. In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above. Thus, under the experimental conditions chosen here, the test substance N-ethylpropylamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP) and the HPRT Test (OECD 476, GLP).

In contrast,the test substance induced micronuclei in vitro in the absence, but not in the presence of S9 (OECD 487, GLP). However, this result could not be confirmed in an in vivo follow-up study (OECD 474, GLP). Under the experimental conditions chosen here, the test substance had no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo . The bioavailability of the test substance in bone marrow after oral administration was clearly demonstrated by effects on the ratio of PCEs and NCEs. Therefore, the in vivo result overrules the in vitro result which is in line with REACH guidance R.7a.

As a result, classification of the test substance for genetic toxicity is not warranted under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.