1-ethylazepan-2-one

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylazepan-2-one

EC Number:

606-384-1

Cas Number:

19797-08-1

Molecular formula:

C8H15NO

IUPAC Name:

1-ethylazepan-2-one

Test material form:

liquid

Method

Target gene:

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

Species / strain

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).

Controls

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.

Results and discussion

Test results

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").

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative