N-acetylhexanelactam

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In order to evaluate the mutagenic potential of the test item registered in bacteria, two Bacterial Reverse Mutation tests are available. Based on the results of both the studies, it can be concluded that the test item registered, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test condition.

In order to evaluate the mutagenic potential of test item registered in mammalian cells, an HGPRT Test is available. Based on the results obtained in this In vitro mammalian cell gene mutation test, the test item registered is considered non-mutagenic at and up to the concentration of 2 mg/mL both in the presence and absence of metabolic activation under the laboratory conditions tested.

In order to evaluate the clastogenic potential of test item registered an in vitro chromosome aberration test in cultured Chinese Hamster Ovary (CHO-K1) cells is available. Based on the results obtained, the test item registered is considered as non-clastogenic up to the concentration of 2 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-10-27 to 1998-11-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study is performed according to guideline. Only four S. typhimurium strains were tested as required at the time of testing. Acceptable, well documented study report which meets basic scientific principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: S. typhimurium TA 97a, TA 98, TA 100, TA 1535

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate from phenobarbital (i.p.) and ß-naphthoflavone(oaral) induced rats (S9)

Test concentrations with justification for top dose:

Experiment 1.: 0.005, 0.05 and 0.5 (mg/plate)
Experiment 2.: 0.05, 0.16, 0.5, 1.6 and 5.0 (mg/plate)

Vehicle / solvent:

bidistilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-amino-anthracene, nitrofurantoine; 4-nitro-1, 2-phenylene-diamine; ICR 191;

Details on test system and experimental conditions:

Three replicates per concentration level and control. Duration of incubation time of each experiment was 48 hours. The first study was conducted with 3 concentration levels of the test substance (0.005 - 0.5 mg/plate). Based on the results of the first experiment the second experiment was performed with 5 concentration levels (0.05-5.0 mg/plate) with and without metabolic activation.

Evaluation criteria:

Since the reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn were not included into evaluation processes.

Statistics:

Arthmetic mean values and standard deviations were calculated out of colonies per plate of three replicates. For evaluation of the results the induction rate the mean values were calculated:
Induction rate = revertant colonies of test substance divided by revertant colonies of the corresponding control.
The test substance is to interpret mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.
Calculations were performed using software Excel 5.0, Microsoft corporation.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

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

Based on the results of this test the test substance is regarded to be not mutagenic.

Executive summary:

In this study (Ames test) Acetylcaprolactam was found to have no mutagenic effects onSalmonella typhimuriumstrains TA 97a, TA 98, TA 100 and TA 1535 with and without metabolic activation system at concentrations of 0.005 - 5.0 mg/plate. No cytotoxic effects were observed at these dose levels.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Iberica Production, S.A., Tarragona Site, 43110 La Canonja, Spain

- Batch No.of test material: ESD0024613
- Expiration date of the batch: After 06 May 2018
- Purity as per Certificate of Analysis: Min. 95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+21 to +29°C)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108 USA
- Suitability of cells: Test approaches currently accepted under the OECD guidance for the assessment of mammalian cell chromosome damage involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the chromosome damage by a variety of chemicals.
An established CHO cell line is useful in in vitro cytogenetics testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time.

- Cell cycle length, doubling time or proliferation index: Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a doubling time approximately 12 to 14 hours and modal chromosome number of 20 was used as the test system.
The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air).

- Methods for maintenance in cell culture if applicable: Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics (penicillin, streptomycin and amphotericin), L-glutamine and 5 or 10% fetal bovine serum (F-12 FBS 5/10).
Dulbecco’s Phosphate buffered saline (PBS)
Trypsin: EDTA solution
Colchicine

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat), 5 days prior to sacrifice. S9 homogenate was stored in a deep freezer maintained at -68 to -86 ºC.

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item were selected for testing in the chromosomal aberration assay:

Experiment No. Test Concentrations
Experiments 1 & 2 a) 5 b) 100 and c) 2000 µg/mL (factor of 20)

Experiment 3 d) 4 e) 20 and f) 100 µg/mL (factor of 5)

Vehicle / solvent:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Exponentially growing CHO-K1 cells were plated at a density of approximately 106 cells in 75 cm2 flask in duplicate with 15 mL F-12 FBS10 and incubated for approximately 24 hours at 37 ± 1oC in a humidified atmosphere of 5 ± 0.2 % CO2 in air.
At the time of preparation of target cells, two parallel cultures were kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of RICC.

- Cell density at seeding (if applicable): 106 cells in 75 cm2

STAIN (for cytogenetic assays): The slides were stained with freshly prepared Giemsa stain in water, for 120 minutes, washed in water, air dried, immersed in xylene and mounted with DPX. The slides were then coded by an individual not involved in scoring process before evaluation

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: Metaphases of the different concentrations of the test items, the positive and vehicle control cultures were scored.

Three hundred metaphases whose centromere number varies not more than by ± 2 from the modal number of 20 were evaluated among the 2 replicates. The chromosome number was recorded for all cells analyzed and the vernier readings of only aberrant cells were recorded. Aberrations, if any were recorded as chromatid / chromosome gaps or breaks and exchange figures. Since gaps are not considered as true aberration, the results are presented as metaphases with aberrations including gaps and excluding gaps.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

• At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
• The increase is dose-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Summary Results of Chromosomal Aberration Assay - Experiment 1 (Presence of Metabolic Activation, 3-hour Exposure)

Refer Appendices 3 and 4

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO	300	2 (0.7)	0	0	0	0	0	0	2 (0.7)	0	0
5	300	2 (0.7)	1 (0.3)	0	0	0	0	0	3 (1.0)	0	13
100	300	1 (0.3)	1 (0.3)	0	0	0	0	0	2 (0.7)	0	36
2000	300	1 (0.3)	1 (0.3)	0	1	0	0	0	3 (1.0)	1 (0.3)	57
CPA 55	300	16 (5.3)	33 (11.0)	70 (23.3)	123 (41.0)	44 (14.7)	21 (7.0)	0	167  (55.7)	+ 167 (55.7)	24

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  ; Cs: Chromosome type;   Ct: Chromatid type               RC: Ring chromosome    

CPA: Cyclophosphamide monohydrate          +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

SummaryResults of Chromosomal Aberration Assay - Experiment 2 (Absence of Metabolic Activation, 3-hour Exposure)

Refer Appendices 3 and 5

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO	300	0	0	0	0	0	0	0	0	0	0
5	300	1 (0.3)	1 (0.3)	0	0	0	0	0	2 (0.7)	0	11
100	300	0	0	0	0	0	0	0	0	0	34
2000	300	2 (0.7)	1 (0.3)	0	0	0	0	0	3 (1.0)	0	52

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  ; Cs: Chromosome type; Ct: Chromatid type; RC: Ring chromosome              

Note: There were no incidences of polyploidy and endoreduplicated cells

Summary Results of Chromosomal Aberration Assay - Experiment 3 (Absence of Metabolic Activation, 21-hour Exposure)

Refer Appendices 3 and 6

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
DMSO	300	0	0	0	0	0	0	0	0	0	0
4	300	0	0	0	0	0	0	0	0	0	17
20	300	0	0	0	0	0	0	0	0	0	37
100	300	0	1 (0.3)	0	1 (0.3)	0	0	0	2 (0.7)	1	53
EMS 600	300	0	0	5 (1.7)	64 (21.3)	74 (24.7)	26 (8.7)	0	161 (53.7)	+ 161 (53.7)	32

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  ; Cs: Chromosome type;  Ct: Chromatid type; RC: Ring chromosome    

EMS: Ethyl methanesulfonate          +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

Conclusions:

The test item, N-acetylhexanelactam was not clastogenic in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The clastogenic potential of the test item,N-acetylhexanelactamto induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

The test item was soluble in dimethyl sulfoxide (DMSO)at 200 mg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, exhibited required level of cytotoxicity as RICC at the highest tested concentration of 2000 µg/mL, compared to the DMSO control, both in the presence and absence of metabolic activation with 3-hour exposure. In the absence of metabolic activation with 21-hour exposure, required level of cytotoxicity was observed between 50 and 128 µg/mL compared to the DMSO. The test item did not precipitate in the test medium and did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 2000 µg/mL in experiments 1 and 2 and a maximum of 100 µg/mL in experiment 3 of the chromosomal aberration assay were tested.

In the definitive chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for 3 hours at concentrations of 5, 100 and 2000 µg/mL in experiments 1 and 2 (presence and in the absence of metabolic activation, respectively). Similarly, in experiment 3, CHO-K1 cells were exposed to the test item in duplicate for 21 hours at concentrations of 4, 20 and 100 µg/mL in the absence of metabolic activation. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

At the respective highest concentrations tested, the reduction in cell growth as RICC was 57, 52 and 53 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

A total of 300 metaphases each from the DMSO control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item, N-acetylhexanelactam was not clastogenic at the concentrations tested and under the conditions of testing.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant
- Batch No.of test material: ESD0024613
- Expiration date of the lot/batch: After 06 May 2018
- Purity as per Certificate of Analysis: Min. 95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+21 to +29°C)
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in Sterile water at 50,000 µg/mL

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Salmonella typhimurium: Health Protection Agency, National Collection of Type Cultures (NCTC), 61, Colindale Avenue, London NW9 5EQ, Great Britain

Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system.

Test concentrations with justification for top dose:

Test concentration are: 50, 158, 500, 1581 and 5000 µg per plate.

N-Acetylhexanelactam did not show toxicity at any of the tested doses either in the presence or in the absence of metabolic activation as the intensity of the bacterial background lawn as well as the mean number of revertant colonies was comparable to the vehicle control plates.

Based on these observations, the test item was tested up to the regulatory-required top dose of 5000 µg/plate in the mutation assay.

Vehicle / solvent:

One hundred microliters (100 µL) of SW was used as the vehicle control.

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 67 hours
- Plate incorporation: Exposure duration is 67 hours

NUMBER OF REPLICATIONS: 3 replicates per group per strain

DETERMINATION OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: Reduction in background lawn

Rationale for test conditions:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

• Tester strain integrity

All Salmonella typhimurium tester strains and the Escherichia coli tester strain used in this assay must demonstrate their growth potential in the presence of histidine and tryptophan, respectively.

All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation, respectively.

Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation.

Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.

• The spontaneous reversion rates in the vehicle control must be in the range of in-house historical data.
• There must be at least three non-toxic dose levels.
• The top dose selected should demonstrate toxicity. In case of non-toxic test items, the top dose tested should be 5000 µg/plate.
• All tester strain culture titers must be in the range of 1-2 x 109 cells/mL to ensure that appropriate numbers of bacteria are used for plating.
• The positive control substances should produce a more than 3-fold increase in mutant colony frequencies when compared to the respective vehicle control plates.

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

TABLE 1: Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

   

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
SW	26	3	NA	112	10	NA	13	2	NA	12	1	NA	146	8	NA
50	25	3	0.96	111	6	0.99	12	2	0.92	10	2	0.83	144	9	0.99
158	24	3	0.92	107	8	0.96	14	2	1.08	11	1	0.92	144	8	0.99
500	26	2	1.00	112	6	1.00	13	1	1.00	10	2	0.83	144	6	0.99
1581	26	3	1.00	104	10	0.93	13	2	1.00	11	1	0.92	142	6	0.97
5000	26	2	1.00	113	3	1.01	14	2	1.08	11	2	0.92	146	6	1.00
Positive controls	570^c	13^c	21.92^c	880^c	18^c	7.86^c	172^c	19^c	13.23^c	164^c	8^c	13.67^c	575^d	21^d	3.94^d
^aValues are means of three replicates calculated from Appendix 2 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate); ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                          ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate); NA: Not applicable; SD: Standard deviation

TABLE 2: Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
SW	25	2	NA	114	4	NA	14	2	NA	11	1	NA	144	6	NA
50	26	2	1.04	107	11	0.94	14	1	1.00	10	1	0.91	142	9	0.99
158	26	3	1.04	108	8	0.95	13	2	0.93	10	2	0.91	143	10	0.99
500	25	2	1.00	104	14	0.91	14	2	1.00	12	2	1.09	143	3	0.99
1581	25	3	1.00	106	6	0.93	13	2	0.93	11	2	1.00	141	8	0.98
5000	25	1	1.00	111	9	0.97	13	1	0.93	11	1	1.00	146	5	1.01
Positive controls	273^c	16^c	10.92^c	580^d	20^d	5.09^d	175^d	14^d	12.50^d	151^e	9^e	13.73^e	566^f	20^f	3.93^f
^aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number  ^bRatio of treated/vehicle control (mean revertants per plate); ^cTA98: 2-Nitrofluorene (2 µg/plate); ^dTA100, TA1535: Sodium azide (1 µg/plate),            ^eTA1537: 9-Aminoacridine (50 µg/plate);  ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)           NA: Not applicable;  SD: Standard deviation

TABLE 3: Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
SW	26	3	NA	110	10	NA	14	1	NA	10	2	NA	146	8	NA
50	25	2	0.96	120	8	1.09	13	1	0.93	11	2	1.10	138	2	0.95
158	25	3	0.96	115	9	1.05	14	2	1.00	11	2	1.10	145	6	0.99
500	26	2	1.00	116	13	1.05	14	2	1.00	11	2	1.10	144	6	0.99
1581	24	3	0.92	105	8	0.95	14	2	1.00	10	2	1.00	146	6	1.00
5000	24	1	0.92	111	9	1.01	13	1	0.93	10	2	1.00	141	6	0.97
Positive controls	568^c	21^c	21.85^c	878^c	19^c	7.98^c	167^c	19^c	11.93^c	169^c	16^c	16.90^c	575^d	16^d	3.94^d
^aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number          ^bRatio of treated/vehicle control (mean revertants per plate); ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate); NA: Not applicable; SD: Standard deviation

TABLE 4: Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
SW	26	3	NA	110	8	NA	14	2	NA	11	1	NA	144	10	NA
50	25	2	0.96	111	10	1.01	13	1	0.93	10	2	0.91	138	3	0.96
158	23	1	0.88	105	7	0.95	14	1	1.00	9	1	0.82	146	5	1.01
500	26	3	1.00	104	7	0.95	13	2	0.93	11	1	1.00	144	7	1.00
1581	24	1	0.92	107	8	0.97	13	1	0.93	11	1	1.00	141	9	0.98
5000	24	2	0.92	100	4	0.91	13	1	0.93	10	1	0.91	142	6	0.99
Positive controls	267^c	17^c	10.27^c	572^d	11^d	5.20^d	173^d	12^d	12.36^d	175^e	6^e	15.91^e	563^f	12^f	3.91^f
^aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate); ^cTA98: 2-Nitrofluorene (2 µg/plate); ^dTA100, TA1535: Sodium azide (1 µg/plate)             ^eTA1537: 9-Aminoacridine (50 µg/plate); ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)             NA: Not applicable; SD: Standard deviation

Conclusions:

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, N-Acetylhexanelactam, was not mutagenic in this bacterial reverse mutation test up to the regulatory-required top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

The test item, N-Acetylhexanelactam was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

N-Acetylhexanelactam is soluble in sterile water (SW) at 50,000 µg/mL.

 

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test itemdid not precipitate on the basal agar plates at any of the tested doses.The test item did not show toxicity to the tester strain TA100 at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the test item was tested up to the regulatory-required top dose of 5000 µg/plate in the mutation assay.

 

In the mutation assay, the bacterial tester strains were exposed to N-Acetylhexanelactamin triplicate at 50, 158, 500, 1581 and 5000 mg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure and the confirmatory mutation assay was conducted using the pre-incubation mode of exposure. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

 

The results of the study from both the initial and confirmatory mutation assay showed that, N-Acetylhexanelactam did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

 

The study indicated that N-Acetylhexanelactam was not mutagenic in this Bacterial Reverse Mutation Assay up to the regulatory-required top dose of 5000 µg/plate, under the conditions of testing employed.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian cell gene mutation test using the Hprt Gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Iberica Production, S.A., Tarragona Site, 43110 La Canonja, Spain
- Batch No.of test material: ESD0024613
- Expiration date of the batch: After 06 May 2018
- Purity as per Certificate of Analysis: Min. 95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+21 to +29°C)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108 USA
- Suitability of cells: Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time.
- Cell cycle length, doubling time or proliferation index: Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 12 to 14 hours was used.

This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.

The cell line was screened for the absence of mycoplasma contamination and was certified free of mycoplasma contamination on 12 January 2017. The karyotype analysis of this cell line is periodically performed by an external laboratory and the modal chromosome number is 20.

Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air).
- Methods for maintenance in cell culture if applicable: Stock cultures of the CHO-K1 cell line were stored at the test facility as frozen permanents in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.

Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.

Basic medium supplemented with 5% fetal bovine serum (FBS) was the treatment medium and was used for target cell exposure to the test item and controls.

Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.

Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.

Dulbecco’s Phosphate buffered saline (PBS) (pH: 7.4)
Trypsin: EDTA solution

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes)

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. Batches are stored in a deep freezer at -68 to -86 ºC.

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in Experiments 1 and 2 (presence and absence of metabolic activation, respectively) of the gene mutation test:

A) 74 B) 222 C) 667 and D) 2000 µg/mL (factor of 3)

Vehicle / solvent:

One hundred fifty microliters (150 L) of DMSO was used per 15 mL of treatment medium as the vehicle control in each of the experiments.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Exponentially growing CHO-KI cells were plated in two replicates and each replicate has two flasks containing 15 mL of complete medium at a density of approximately 4.5 x 106 cells / 75 cm2 flask and incubated for approximately 24 hours.

Two parallel cultures were also kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of Relative Survival (RS).

- Cell density at seeding (if applicable): 4.5 x 106 cells / 75 cm2

DURATION
All test item and positive control concentrations were prepared immediately before use in sterile tubes. The target cells were exposed to the vehicle, positive control and various concentrations of the test item for 3 hours in the presence and absence of metabolic activation.

The medium from each target cells flask was removed by aspiration and replaced with 13.5 mL and 15 mL of treatment medium for the experiment in the presence and absence of metabolic activation, respectively. For the experiment incorporating metabolic activation, 1.5 mL of S9 mix was added to give a final concentration of 1 % S9 (v/v) in the test medium.

One hundred fifty microliters (150 µL) each of the vehicle control, the positive control, dilution of the test item were transferred to respective flasks and gently mixed and kept for incubation to expose the cells to treatment.

SELECTION AGENT (mutation assays): Replicate cultures from controls and each treatment level were trypsinized, and the cells suspended in 10 mL complete medium, pooled, and counted using a hemocytometer.

For selection of the 6-Thioguanine (6-TG) resistant phenotype, cells from each of the replicate cultures were plated in to 5 flasks at a density of approximately 2 x 105 cells/25 cm2 flask (total of 106 cells/replicate) in selective medium and incubated for 10 days.

For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated approximately at 200 cells/25 cm2 flask in triplicate in cloning medium and incubated for 10 days.

STAIN (for cytogenetic assays): The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.

NUMBER OF REPLICATIONS: Two

Evaluation criteria:

When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	2	0	3	1	2	16	0.0000080	182	178	180	0.91	8.79
	1	2	2	0	3			179	189	184
74	2	1	3	0	2	14	0.000007	167	158	182	0.84	8.33
	1	2	0	2	1			159	170	169
222	0	2	1	2	1	13	0.000007	166	179	150	0.81	8.02
	3	0	1	1	2			151	157	164
667	0	1	2	2	0	12	0.000006	169	160	170	0.83	7.23
	2	3	2	0	0			160	167	165
2000	3	2	1	0	1	14	0.000007	157	160	155	0.79	8.86
	2	0	1	2	2			162	152	160
3-MCA	34	40	42	35	30	360	0.000180	170	167	161	0.82	219.51^+
	36	35	37	37	34			165	159	160

Positive Control: 8 µg/mL 3-MCA             Vehicle Control: DMSO                                                 CE: Cloning Efficiency               MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

⁺: Mutant frequency of 6-TG mutants is significantly higher than the concurrent vehicle control value (p < 0.05)

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

 

 

 

Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	1	2	0	0	2	11	0.0000055	180	184	178	0.90	6.11
	3	1	2	0	0			185	181	176
74	2	3	1	1	2	14	0.000007	178	180	169	0.87	8.05
	0	0	2	0	3			170	175	171
222	1	2	0	3	1	13	0.000007	164	171	168	0.84	7.74
	2	2	1	0	1			166	163	172
667	3	3	1	0	0	13	0.000007	165	160	158	0.80	8.13
	2	0	1	2	1			164	155	161
2000	4	1	2	0	0	11	0.000006	159	164	167	0.80	6.88
	1	0	0	2	1			161	157	154

Vehicle Control: DMSO                             CE: Cloning Efficiency                    MF: Mutant Frequency                                                              

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

 

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

 

 

 

 

 

 

Conclusions:

The test item, N-acetylhexanelactam does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The genotoxic potential of the test item N-acetylhexanelactam to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation assay. The gene mutation assay comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

N-acetylhexanelactam was soluble in dimethyl sulfoxide (DMSO) at 200 mg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant growth inhibition as Relative Survival at any of the tested concentrations either in the presence or in the absence of metabolic activation. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 2000 µg/mL was tested in the gene mutation assay. 

In the gene mutation assay, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 74, 222, 667 and 2000 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical condition

The results of the forward gene mutation test at the hprt locus with N-acetylhexanelactam indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In order to evaluate the genetic toxicity potential of the registered substance four in vitro tests according to OECD guidelines were performed: i.e. two Ames tests (OECD 471), Chromosome aberration test (OECD473) and HGPRT (OECD 476) test. All of these four in vitro tests were negative in result. According to Regulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, the test substance N-acetylhexanelactam has not to be classified.