1-ethylazepan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 490, Thymidine kinase test): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 16 Apr 2018

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:

adopted Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted May 2008

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium strains) and trp operon (E. coli strains)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

The dose-range finding test served as Experiment 1.
Experiment 1: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Experiment 2: 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test item was miscible in Milli-Q water at 25 °C and stable for at least 24 h at room temperature under normal laboratory light conditions, for at least 8 days in refrigerator (2-8°C) and for at least 3 weeks in the freezer (≤ -15°C), which was confirmed over the concentration range 1 to 60 mg/mL (project # 20144290 at the same testing facility).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: -S9: 4-nitroquinoline N-oxide 10 µg/plate for WP2uvrA and ICR-191 2.5 µg/plate for TA1537 (Exp. 1); +S9: 2-aminoanthracene 1, 2.5 or 15 µg/plate for all strains

Remarks:

Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 30 ± 2 min (Experiment 2)
- Exposure duration: 48 ± 4 h (Experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicates each in 2 independant experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not precipitate in the incubation medium at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES: Experiment 1 served as range-finding study.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data (please refer to table 4 under "any other information on results incl. tables").

Table 1: Results of dose-range finding study/Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
Without S9-mix	TA100	WP2uvrA
Positive control	1040± 42	1093 ± 98
Solvent control	123 ± 13	36 ± 8
1.7	123 ± 4	31 ± 7
5.4	132 ± 18	26 ± 6
17	117 ± 5	36 ± 5
52	105 ± 7	34 ± 4
164	124 ± 23	32 ± 8
512	123 ± 12	41 ± 8
1600	123 ± 27	44 ± 6
5000	121 ± 8	33 ± 6
With S9-mix	TA100	WP2uvrA
Positive control	1513 ± 3	401 ± 118
Solvent control	141 ± 12	40 ± 4
1.7	128 ± 14	41 ± 8
5.4	122 ± 13	45 ± 4
17	108 ± 11	47 ± 7
52	110 ± 16	36 ± 6
164	114 ± 13	32 ± 6
512	103 ± 11	34 ± 9
1600	116 ± 10	36 ± 3
5000	91 ± 8	31 ± 6

Table 2: Results of Experiment 1 (direct plate assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix	TA1535	TA1537	Ta98
Positive control	1073 ± 97	1036 ± 50	1058 ± 40
Solvent control	8 ± 2	3 ± 2	15 ± 4
52	8 ± 1	6 ± 3	16 ± 1
164	12 ± 5	5 ± 5	20 ± 6
512	11 ± 4	4 ± 1	15 ± 6
1600	8 ± 7	6 ± 2	21 ± 2
5000	8 ± 4	7 ± 5	16 ± 6
With S9-mix	TA1535	TA1537	Ta98
Positive control	349 ± 22	471 ± 37	1181 ± 40
Solvent control	10 ± 2	3 ± 2	20 ± 5
52	12 ± 2	11 ± 4	30 ± 11
164	12 ± 7	9 ± 9	26 ± 3
512	14 ± 2	7 ± 0	23 ± 8
1600	7 ± 7	4 ± 5	22 ± 3
5000	10 ± 1	5 ± 2	18 ± 2

Table 3: Results of Experiment 2 (pre-incubation assay)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
Without S9-mix	TA1535	TA1537	TA98	TA100	WP2uvrA
Positive control	1164 ± 79	145 ± 15	1147 ± 80	765 ± 93	217 ± 85
Solvent control	8 ± 4	3 ± 2	18 ± 2	109 ± 15	29 ± 6
52	12 ± 6	3 ± 1	11 ± 4	104 ± 14	32 ± 5
164	11 ± 4	6 ± 3	22 ± 19	105 ± 12	36 ± 8
512	15 ± 4	5 ± 5	17 ± 5	110 ± 17	31 ± 3
1600	17 ± 13	5 ± 2	16 ± 6	106 ± 17	34 ± 6
5000	4 ± 0	5 ± 2	15 ± 5	114 ± 9	39 ±8
With S9-mix
Positive control	283 ± 20	217 ± 21	898 ± 85	1801 ± 423	477 ± 5
Solvent control	21 ± 9	12 ± 2	35 ± 6	110 ± 0	39 ± 8
52	20 ± 5	12 ± 4	32 ± 4	128 ± 14	38 ± 4
164	16 ± 2	18 ± 4	29 ± 2	106 ± 9	43 ± 5
512	19 ± 4	13 ± 4	34 ± 5	106 ± 20	39 ± 8
1600	14 ± 4	15 ± 4	29 ± 6	104 ± 2	46 ± 6
5000	21 ± 1	12 ± 3	27 ± 3	97 ± 13	38 ± 4

Table 4: Historical control data of the positive and negative control

	TA1535		TA1537		TA98		TA100		WP2uvrA
Positive control
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	846	219	787	353	1406	887	901	1232	1094	437
SD	146	119	345	162	258	349	168	343	477	149
n	2348	2229	2003	2234	2200	2276	2335	2327	2021	2085
Negative control
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Apr - 27 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: Blood was collected from three donors with an age of 30, 30 and 32 years, respectively. Average generation time was 14.0, 14.8 and 14.8 h, respectively.
- Whether whole blood or separated lymphocytes were used: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Methods for maintenance in cell culture: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 54 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.6 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment.

MEDIA USED
- Type and identity of media: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) and 30 U/mL heparin.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

Dose-range finding test:
3, 24 and 48 h treatment without metabolic activation, respectively: 62.5, 125, 250, 500, 1000 and 1412 µg/mL

Main experiment:
3 h treatment with and without metabolic activation: 500, 1000 and 1412 µg/mL
24 h treatment without metabolic activation: 10, 1000 and 1412 µg/mL
48 h treatment without metabolic activation: 10, 750 and 1200 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48 h
- Expression time (cells in growth medium): 3 h treatment: 20 - 22 h
- Fixation time: 3 h treatment: 24 h; 24 and 48 h treatment: 24 and 48 h, respectively

SPINDLE INHIBITOR: Colchicine (0.5 μg/mL medium)

STAIN: 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: duplicate cultures each in 2 independent experiments

NUMBER OF CELLS EVALUATED: 300 (150 cells per culture)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 75

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

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:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A concentration of 0.01 M (highest tested dose level, 1412 μg/mL) showed no precipitation in the culture medium.

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test blood cultures were treated with 62.5, 125, 250, 500, 1000 and 1412 μg N-ethylcaprolactam/mL culture medium for 3 h with and without S9-mix, and for 24 and 48 h without S9 mix, respectively. The test item did not show cytotoxic properties in the short-term experiment of 3 h with and without metabolic activation system. In the long-term experiment a decrease of the mitotic index was observed at the highest tested dose level of 1412 µg/mL after 24 and the 48 h.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive controls both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.

Table 1: Dose-range finding study

Concentration (µg/mL)	Number of metaphases
	Absolute	Number of cells scored	% control
Without metabolic activation (-S9-mix) 3 h exposure time, 24 h fixation time
Control a)	82	1002	100
62.5	88	1024	107
125	76	1017	93
250	78	1012	95
500	72	1014	88
1000	85	1006	104
1412	64	1010	78
Without metabolic activation (-S9-mix) 24 h exposure time, 24 h fixation time
Control a)	73	1014	100
62.5	48	1016	66
125	56	1012	77
250	34	1009	47
500	40	1011	55
1000	39	1003	53
1412	18	1005	25
Without metabolic activation (-S9-mix) 48 h exposure time, 48 h fixation time
Control a)	63	1013	100
62.5	47	1018	75
125	45	1013	71
250	40	1011	63
500	41	1003	65
1000	34	1022	54
1412	17	1016	27
With metabolic activation (+S9-mix) 3 h exposure time, 24 h fixation time
Control a)	85	1013	100
62.5	78	1021	92
125	66	1017	78
250	69	1009	81
500	71	1009	84
1000	51	1007	60
1412	61	1012	72

a) RPMI 1640 medium

Table 2: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

	RPMI-C			500 µg/mL			1000 µg/mL			1412 µg/mL			0.5 µg/mL MMC-C
Culture	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
Mitotic Index (%)	100			92			87			80			62
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps) a)	2	4	6	1	2	3	3	0	3	0	2	2	39	25	64***
No. of Cells with aberrations (- gaps)	2	4	6	1	2	3	3	0	3	0	2	2	39	25	64***
Chromatid break	2	4			2		1				2		34	17
Chromosome break				2			2						2	3
Exchange figure													8	6
Miscellanous							2 endo							3 intra	intra
total aberrations (+ gaps)	2	4		2	2		3	0		0	2		47	27
total aberrations (- gap)	2	4		2	2		3	0		0	2		47	27

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

endo: endoreduplication

intra: chromosome intrachange

Table 3: Chromosome aberrations in human lymphocyte cultures treated with the test item in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

	RPMI-C			500 µg/mL			1000 µg/mL			1412 µg/mL			10 µg/mL CP
	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
Mitotic Index (%)	100			90			81			77			48
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps) a)	2	2	4	1	2	3	3	3	6	2	0	2	26	30	56***
No. of Cells with aberrations (- gaps)	2	2	4	1	2	3	3	3	6	2	0	2	26	30	56***
Chromatid break	1	2		1	2		3	1		1			18	38
Chromosome break	1							1					8	7
Exchange figute								1		1			3	4
Miscellanous							endo	2 endo			endo		intra
total aberr (+ gaps)	2	2		1	2		3	3		2	0		30	49
total aberr (- gaps)	2	2		1	2		3	3		2	0		30	49

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

CP: Cylcophosphamide

endo: endoreduplication

intra: chromosome intrachange

Table 4: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

	RPMI-C			10 µg/mL			1000 µg/mL			1412 µg/mL			0.2 µg/mL MMC-C
Culture	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
Mitotic Index (%)	100			95			61			47			34
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps) a)	1	0	1	1	0	1	0	0	0	0	0	0	24	24	48***
No. of Cells with aberrations (- gaps)	1	0	1	1	0	1	0	0	0	0	0	0	23	24	48***
Chromatid break	1			1									11	9
Chromosome break													5	7
Exchange figure													7	9
Dicentric chromosome														1
Miscellanous							poly			poly			p
total aberr (+ gaps)	1	0		1	0		0	0		0	0		25	26
total aberr (- gaps)	1	0		1	0		0	0		0	0		24	26

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

p: pulverized chromosome

poly: polyploidy

Table 5: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc	RPMI-C			10 µg/mL			750 µg/mL			1200 µg/mL			0.1 µg/mL MMC-C
Culture	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
Mitotic Index (%)	100			107			65			50			50
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+ gaps) a)	1	0	1	1	0	1	0	2	2	0	1	1	29	34	63***
No. of Cells with aberrations (- gaps)	1	0	1	0	0	0	0	2	2	0	0	0	27	34	61***
Chromatid gap				1							1		1
Chromsome gap													1
Chromatid break								1					8	21
Chromosome break								1					9	5
Exchange figure													13	8
chromatid deletion	1
Miscellanous													p	p
total aberrations (+ gaps)	1	0		1	0		0	2		0	1		32	35
total aberrations (- gaps)	1	0		0	0		0	2		0	0		30	35

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

MMC-C: Mitomycin C

p: pulverized chromosome

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug - 08 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted Jul 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Suitability of cells: recommended test system in international guidelines
- Methods for maintenance in cell culture: Cells were cultured in a humid atmosphere (actual range 77 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.5°C).

MEDIA USED
- Type and identity of media: Basic medium was a RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. Basis medium supplemented with 5, 10 or 20% heat-inactivated horse medium was used as exposure, growth, selective and non-selective medium, respectively.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding study
With and without S9 mix: 125, 250, 500, 1000 and 1412 µg/mL (3 h)
Without S9 mix: 125, 250, 500, 1000 and 1412 µg/mL (24 h)

Experiment I
With and without S9 mix: 16, 31, 63, 125, 250, 500, 1000 and 1412 µg/mL (3 h)

Experiment II
Without S9 mix: 62.5, 125, 250, 500, 750, 1000, 1250 and 1412 µg/mL (24 h)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 2 days
- Selection time: 11 or 12 days

SELECTION AGENT (mutation assays):
5 μg/mL trifluorothymidine

STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
Duplicate measurements each of 5 wells for mutant frequency and duplicate measurements each of 2 wells for cloning efficiency, respectively, in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro-well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 at a concentration of 1412 μg/mL was 8.11 compared to 8.10 in the solvent control.
- Effects of osmolality: The osmolarity at a concentration of 1412 μg/mL was 0.299 Osm/kg compared to 0.288 Osm/kg in the solvent control.
- Precipitation: The test substance did not precipitate in the exposure medium up to and including the highest concentration of 1412 μg/mL (= 10 mM).

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 125 to 1412 μg/mL in the absence of S9-mix with 3 and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period. After 3 h of exposure, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1412 μg/mL compared to the suspension growth of the solvent control. After 24 h of exposure, the relative suspension growth was 35% at the test item concentration of 1412 μg/mL compared to the relative suspension growth of the solvent control.

HISTORICAL CONTROL DATA
- Positive historical control data: The mutation frequencies of both positive controls were within the range of the historical control data (please refer to Table 3 under 'any other information on results incl. tables).
- Negative (solvent/vehicle) historical control data: The mutation frequency of the solvent control was within the range of the historical control data (please refer to Table 2 under 'any other information on results incl. tables).

Table 1: Results of Experiment 1 (3 h incubation) with and without metabolic activation and Experiment 2 (24 h incubation) without metabolic activation

dose	RSG	CE day2	RCE	RTG	mutation frequency per 10^6survivors
(µg/ml)	(%)	(%)	(%)	(%)	total	small	large
without metabolic activation: 3 h treatment
SC1	100	86	100	100	60	11	48
SC2		84			82	21	59
16	107	76	89	95	76	22	51
31	99	107	126	124	61	13	47
63	97	88	103	100	58	23	33
125	101	90	106	107	69	15	52
250	104	91	107	112	78	24	51
500	115	93	109	126	89	26	59
1000	108	98	115	124	78	21	55
1412	104	101	119	123	55	20	33
MMS	86	57	67	57	630	427	158
SC1	100	105	100	100	76	27	46
SC2		88			76	33	41
16	109	88	91	99	82	13	67
3	97	113	117	13	74	18	54
63	84	99	103	87	70	26	41
125	98	108	112	110	73	22	49
250	111	86	90	100	91	26	62
5000	74	150	155	115	65	18	45
1000	100	86	90	89	64	31	31
1412	91	131	135	123	64	20	41
CP	69	99	103	71	476	288	138
without metabolic activation: 24 h treatment
SC1	100	81	100	100	118	82	31
SC2		97			88	65	20
16	106	95	107	113	109	71	33
31	118	90	101	120	103	60	38
63	127	85	95	121	122	70	46
125	91	89	100	91	107	67	35
250	64	94	106	86	116	79	32
500	46	90	101	47	94	69	22
1000	32	79	89	29	109	74	31
1272	31	125	140	43	89	63	22
MMS	75	68	77	57	829	411	304

CP: Cylcophosphamide

MMS: Methylmethanesulfonate

Table 2: Historical control data of the solvent control

	Mutation frequency per 10E+6 survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	101	98	100
SD	30	31	30
n	279	262	293
Upper control limit (95% control limits)	170	162	165
Lower control limit (95% control limits)	31	34	36

Table 3: Historical control data of the positive control

	Mutation frequency per 10E+6 survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	803	695	1545
SD	253	223	887
n	142	132	151
Upper control limit (95% control limits)	1533	119	3954
Lower control limit (95% control limits)	72	1270	-864

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria:

N-ethylcaprolactam was tested for its mutagenic potential in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Charles River Laboratories, 2018). Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia colistrain WP2 uvrA were exposed to the test item in the presence and absence of metabolic activation (S9 mix). In two independent experiments, N-ethylcaprolactam was tested at a concentration range of 52 to 5000 µg/plate, using the plate incorporation (first experiment) and the pre-incubation method (second experiment). Vehicle (Milli Q water) and positive controls were included in each experiment. Precipitation of the test substance was not reported and no cytotoxicity occurred up to t The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains either with or without metabolic he highest tested concentration in the presence and absence of S9 mix. activation. Positive and vehicle controls were in the range of historical control data and confirmed the validity of the test system.

Thus, based on the results of the present study and under the experimental conditions chosen, N-ethylcaprolactam is not mutagenic in bacteria with and without metabolic activation.

 

In vitro cytogenicity in mammalian cells:

In vitro cytogenicity in mammalian cells was investigated in a chromosome aberration test performed in accordance with OECD guideline 473 and in compliance with GLP (Charles River Laboratories, 2018). The induction of structural chromosomal damage was evaluated in vitro in cultured peripheral human lymphocytes. In the first experiment, the cells were treated with the test item (vehicle: culture medium) for 3 h at concentrations of 500, 1000 and 1412 µg/mL with and without metabolic activation (S9 mix), and in the second experiment, for 24 h at 10, 1000 and 1412 µg/mL and for 48 h at concentration of 10, 750 and 1200 µg/mL, in the absence of metabolic activation. For the 48 h exposure time, appropriate toxicity was reached at this dose levels. Vehicle control and positive control substances (mitomycin C and cyclophosphamide) were included. All control substances showed the expected results and were within the range of historical control data, thus demonstrating the validity of the test system and the functionality of the S9 mix.

N-ethylcaprolactam did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome abberations in the absence and presence of S9 mix. In addition, the frequency of polyploid cells and cells with endoreduplicated chromosomes were not affected with or without metabolic activation.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in the chromosomal aberration test with and without metabolic activation in human lymphocytes in vitro.

 

In vitro gene mutation in mammalian cells:

N-ethylcaprolactam was tested for its mutagenic potential in mammalian cells according to OECD guideline 490 and in compliance with GLP (Charles River Laboratories, 2018). Two independent experiments were performed in L5178Y mouse lymphoma cells (thymidine kinase locus). The cells were treated with the test item at eight concentrations up to 1412 µg/mL for 3 h (first experiment) with and without metabolic activation (S9 mix) and for 24 h (second experiment) in the absence of metabolic activation. No toxicity was observed in the first experiment at the top concentration. In the second experiment the relative total growth was reduced to 43% at the test item concentration of 1412 µg/mL. Concurrent vehicle controls (water) and positive control substances were included. The mutation frequency of the vehicle control was within the range of historical control data. The positive control substances cyclophosphamide (+S9 mix) and methylmethanesulfonate (–S9 mix) significantly increased the mutation frequency in L5178Y cells, thus demonstrating the sensitivity and validity of the test system.

Under the conditions of the test, the N-ethylcaprolactam did not induce a biologically relevant increase in mutation frequency in any of the tested concentrations, either in the presence or absence of metabolic activation. Thus, the test item was not considered to be mutagenic in mammalian cells.

 

Conclusion:

In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that N-ethylcaprolactam is neither mutagenic nor clastogenic in vitro.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.