N-acetylhexanelactam

Endpoint summary

Description of key information

In order to evaluate the skin sensitizing property of the test substance registered three EURL ECVAM validated in vitro tests were performed: DPRA (OECD 442C), h-CLAT (OECD 442E) and ARE.Nrf2 -Luciferase test (Keratinosens)(OECD 442D).

The first one, i.e. DPRA (OECD 442C), resulted in mean depletion of both peptides Lysine and Cysteine of 11.75 % and thus was greater than 6.38 % (acc. to Model 1, OECD 442C). In this study under the given conditions the test item showed low reactivity towards the peptides and is considered to potentially have a sensitizing property. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Under the given conditions in a second in vitro test, i.e. h-CLAT (OECD 442E) the test item did not upregulate the expression of the cell surface marker in at least two independent experimental runs and is considered not sensitizing. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

In a third in vitro test, i.e. Keratino Sens, ARE-Nrf2 Luciferase OECD 442D), under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experimental runs. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

As two out of three in vitro tests were negative in result, the test substance registered is predicted to be a non-sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-27 to 2017-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Specific details on test material used for the study:

Name: N-acetylhexanelactam
Batch No.: ESD0024613
Chemical name: 1-acetylazepan-2-one
CAS No 1888-91-1
Purity 98.1% (GC)
Physical State: liquid
Molecular Weight: 155.2 g/mol
Colour: light yellow clear
Storage Conditions: room temperature
Stability: stable at storage conditions
Expiry Date: 06 May 2018
Safety Precautions: The routine hygienic procedures are sufficient to assure personnel health and safety.

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%] cysteine run

Value:

3.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%] lysine run

Value:

19.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD7	0.0000	0.0000	0.0000	0.0000
STD6	153.8068	0.0167	139.6547	0.0167
STD5	321.6256	0.0334	281.4845	0.0334
STD4	642.2243	0.0667	555.3300	0.0667
STD3	1318.5444	0.1335	1100.1805	0.1335
STD2	2593.4377	0.2670	2176.1533	0.2670
STD1	5075.1016	0.5340	4280.0898	0.5340

 

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1193.1283	0.1239	74.99	74.90	0.25	0.34
	1187.7434	0.1233	75.10
	1210.7732	0.1258	74.62
Test Item	4595.2026	0.4810	3.67	3.82	0.21	5.37
	4592.3970	0.4807	3.73
	4577.0308	0.4791	4.05

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1861.8433	0.2304	55.78	54.93	0.78	1.42
	1926.2322	0.2384	54.25
	1904.2300	0.2357	54.77
Test Item	3385.4136	0.4205	19.59	19.69	0.81	4.11
	3345.2500	0.4155	20.55
	3413.0500	0.4240	18.93

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model¹

Mean Cysteine andLysine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 prediction model¹

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed low reactivity towards the peptides. The test item is considered to potentially have a sensitizing property but the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study N-acetylhexanelactam was dissolved in acetonitrile. Based on a molecular weight of 155.2 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any sample.

No significant co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both peptides by comparing the peptide concentration of the test item samples to the corresponding reference control C (RC C).

In the lysine run a shift of the lysine peptide peak from 7.7 min (STD 1) to 7.0 (REF C2 ACN) up to 6.5 min (REF B6) was observed (see16.4.2). Since the reference control samples allow clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, that indeed the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. Furthermore, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the results.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was >6.38% (11.75%). Based on the prediction model 1 the test item is considered to potentially have a sensitizing property.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-20 to 2017-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”

Version / remarks:

adopted 29 July 2016

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158

Version / remarks:

DB-ALM Protocol n°158, July 1st, 2015

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Name: N-acetylhexanelactam
Batch No.: ESD0024613
Chemical name 1-acetylazepan-2-one
CAS No 1888-91-1
Purity 98.1% (GC)
Physical State: liquid
Molecular Weight: 155.2 g/mol
Colour: light yellow clear
Storage Conditions: room temperature
Stability: stable at storage conditions
Expiry Date : 06 May 2018
Safety Precautions: The routine hygienic procedures are sufficient to assure personnel health and safety.

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (439% experiment 1; 684% experiment 2; 322% experiment 3) and 200% for CD54 (407% experiment 1; 785% experiment 2; 475% experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

118

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

152

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

152

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

152

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

107

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

175

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.1	97.7	97.3	1113	881	745	368	136	84	56	149	118
DMSO Control	0.20%	97.2	97.1	96.8	1140	947	703	437	244	100	100	162	135
DNCB	4.00	89.6	88.8	89.4	2643	1719	726	1917	993	439	407	364	237
N-acetyl-hexanelactam	1000	95.9	96.7	96.0	1188	1036	716	472	320	108	131	166	145
	833.33	96.3	96.7	96.5	1148	1019	716	432	303	99	124	160	142
	694.44	95.4	96.6	96.6	1234	1079	717	517	362	118	148	172	150
	578.70	97.1	96.9	96.5	1149	969	691	458	278	105	114	166	140
	482.25	96.6	96.9	96.8	1086	936	711	375	225	86	92	153	132
	401.88	96.9	97.2	97.0	1155	1085	713	442	372	101	152	162	152
	334.90	96.2	97.2	97.2	1187	1007	754	433	253	99	104	157	134
	279.08	97.1	97.0	97.0	1162	943	724	438	219	100	90	160	130

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.9	97.8	98.0	777	756	584	193	172	79	87	133	129
DMSO Control	0.20%	97.9	97.9	97.4	830	782	585	245	197	100	100	142	134
DNCB	4.0	80.3	79.8	79.6	2365	2237	690	1675	1547	684	785	343	324
N-acetyl-hexanelactam	1000.00	95.9	96.1	96.1	923	815	550	373	265	152	135	168	148
	833.33	96.4	96.3	95.6	900	854	556	344	298	140	151	162	154
	694.44	96.2	96.3	96.2	896	828	562	334	266	136	135	159	147
	578.70	96.0	96.1	96.2	913	801	548	365	253	149	128	167	146
	482.25	96.8	96.6	96.8	891	764	552	339	212	138	108	161	138
	401.88	96.6	96.3	96.6	899	820	534	365	286	149	145	168	154
	334.90	96.8	96.3	97.1	865	810	555	310	255	127	129	156	146
	279.08	96.7	97.0	96.9	907	843	554	353	289	144	147	164	152

CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.4	96.8	97.0	1202	896	647	555	249	75	85	186	138
DMSO Control	0.20%	97.1	97.3	97.2	1355	904	611	744	293	100	100	222	148
DNCB	4.0	84.2	83.8	84.1	3071	2069	677	2394	1392	322	475	454	306
N-acetyl-hexanelactam	1000.0	94.9	94.9	95.0	1286	1102	588	698	514	94	175	219	187
	833.33	96.0	96.1	96.0	1339	976	588	751	388	101	132	228	166
	694.44	95.8	96.3	96.2	1283	1000	596	687	404	92	138	215	168
	578.70	95.7	95.4	96.2	1324	1011	579	745	432	100	147	229	175
	482.25	96.3	95.8	96.4	1308	963	597	711	366	96	125	219	161
	401.88	96.1	96.3	95.8	1400	964	601	799	363	107	124	233	160
	334.90	96.4	96.3	96.3	1318	920	596	722	324	97	111	221	154
	279.08	95.9	96.5	96.7	1369	998	611	758	387	102	132	224	163

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study N-acetylhexanelactam was dissolved in DMSO. For the dose finding assay stock solution with a concentration of 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 could be derived in the dose finding assay.

Based on that result, the main experiment was performed covering the following concentration steps:

1000; 833.33; 694.44, 578.70; 482.25; 401.88; 334.90; 279.08 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 95.9% (CD86), 96.7% (CD54) and 96.0% (isotype IgG1 control) in the first experiment and to 95.9% (CD86), 96.1% (CD54) and 96.1% (isotype IgG1 control) in the second experiment. In the third experiment the relative cell viability at the highest test item concentration was reduced to 94.9% (CD86), 94.9% (CD54) and 95.0% (isotype IgG1 control).

In the first experiment the expression of the cell surface markers CD86 and CD54 was not upregulated above the threshold of 150% and 200%, respectively.

In the second experiment the expression of the cell surface markers CD86 slightly exceeded the threshold of 150% (152%) at the highest test concentration of 1000 µg/mL. In contrast the expression of CD54 was not upregulated above the threshold of 200%.

Due to the equivocal outcome of these two experiments, a third independent run was performed in order to derive a final prediction.

In the third experiment the expression of the cell surface markers CD86 and CD54 was not upregulated above the threshold of 150% and 200%, respectively.

Since the expression of both cell surface markers did not exceeded the thresholds in two independent experiments the test item considered to be no skin sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-09 to 2017-04-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

(adopted: February 04, 2015)

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155,

Version / remarks:

July 1st, 2015

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Name: N-acetylhexanelactam
Batch No.: ESD0024613
Chemical name 1-acetylazepan-2-one
CAS No 1888-91-1
Purity 98.1% (GC)
Physical State: liquid
Molecular Weight: 155.2 g/mol
Colour: light yellow clear
Storage Conditions: room temperature
Stability: stable at storage conditions
Expiry Date: 06 May 2018
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.83 in experiment 1; 2.34 in experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

111.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

119.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	100.4	97.4	98.9	2.1
	8.00	102.2	95.6	98.9	4.6
	16.00	116.3	104.0	110.2	8.7
	32.00	117.7	103.1	110.4	10.3
	64.00	129.4	106.2	117.8	16.4
Test Item	0.98	118.9	119.7	119.3	0.6
	1.95	111.6	101.8	106.7	6.9
	3.91	130.9	114.0	122.5	12.0
	7.81	109.6	121.3	115.5	8.3
	15.63	116.3	126.4	121.3	7.2
	31.25	116.9	121.5	119.2	3.3
	62.50	114.4	114.9	114.7	0.3
	125.00	110.0	114.8	112.4	3.4
	250.00	112.5	119.6	116.0	5.0
	500.00	104.1	105.8	105.0	1.2
	1000.00	103.3	101.6	102.4	1.2
	2000.00	116.8	96.2	106.5	14.6

 

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.20	1.20	1.22	1.21	0.01
	8.00	1.20	1.23	1.29	1.24	0.05
	16.00	1.30	1.25	1.56	1.37	0.17
	32.00	1.86	1.58	2.28	1.90	0.35	*
	64.00	2.86	2.81	2.81	2.83	0.03	*
Test Item	0.98	1.00	0.96	1.14	1.03	0.10
	1.95	1.09	1.08	1.16	1.11	0.04
	3.91	1.20	0.94	0.93	1.02	0.15
	7.81	0.95	1.01	0.98	0.98	0.03
	15.63	0.98	0.83	0.92	0.91	0.07
	31.25	0.99	0.94	1.01	0.98	0.03
	62.50	0.94	0.87	1.13	0.98	0.13
	125.00	1.01	0.97	1.17	1.05	0.11
	250.00	0.89	0.88	1.01	0.93	0.07
	500.00	1.15	0.92	0.94	1.00	0.13
	1000.00	1.08	0.96	0.99	1.01	0.06
	2000.00	1.04	0.99	1.04	1.02	0.03

* = significant induction according to Student’s t-test, p<0.05

 

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.19	1.02	0.99	1.07	0.10
	8.00	1.11	1.04	1.22	1.12	0.09
	16.00	1.25	1.25	1.39	1.30	0.08
	32.00	1.61	1.53	1.69	1.61	0.08	*
	64.00	2.31	2.26	2.47	2.34	0.11	*
Test Item	0.98	0.97	1.11	1.36	1.15	0.20
	1.95	0.97	0.98	0.99	0.98	0.01
	3.91	0.87	0.80	0.83	0.83	0.03
	7.81	0.88	0.99	0.99	0.95	0.07
	15.63	0.89	0.90	0.84	0.88	0.03
	31.25	0.91	0.97	1.01	0.96	0.05
	62.50	0.90	0.92	0.93	0.92	0.01
	125.00	1.06	0.93	1.00	1.00	0.07
	250.00	0.92	0.91	1.13	0.99	0.12
	500.00	0.93	0.94	1.06	0.98	0.07
	1000.00	0.98	1.03	1.12	1.04	0.07
	2000.00	0.99	1.07	1.02	1.03	0.04

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.21	1.07	1.14	0.10
	8.00	1.24	1.12	1.18	0.08
	16.00	1.37	1.30	1.33	0.05
	32.00	1.90	1.61	1.76	0.21	*
	64.00	2.83	2.34	2.59	0.34	*
Test Item	0.98	1.03	1.15	1.09	0.08
	1.95	1.11	0.98	1.05	0.09
	3.91	1.02	0.83	0.93	0.13
	7.81	0.98	0.95	0.97	0.02
	15.63	0.91	0.88	0.90	0.02
	31.25	0.98	0.96	0.97	0.01
	62.50	0.98	0.92	0.95	0.04
	125.00	1.05	1.00	1.02	0.04
	250.00	0.93	0.99	0.96	0.04
	500.00	1.00	0.98	0.99	0.02
	1000.00	1.01	1.04	1.03	0.02
	2000.00	1.02	1.03	1.02	0.00

* = significant induction according to Student’s t-test, p<0.05

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	-	-
Imax	1.11	1.15	1.13	0.03
IC30[µM]	n.a.	n.a.	-	-
IC50[µM]	n.a.	n.a.	-	-

n.a. = not applicable

 

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment2	pass/fail
CV Solvent Control	< 20%	7.9	pass	13.3	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2	pass	2	pass
EC1.5 PC	7 < x < 30 µM	19.95	pass	26.27	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.83	pass	2.34	pass

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study N-acetylhexanelactam was dissolved in DMSO.

Based on a molecular weight of 155.2 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In both experiments no induction of luciferase activity above the threshold value of 1.5 was observed.The maximal luciferase activity induction (I_max) was determined with 1.11 in experiment 1 and 1.15 inexperiment 2. Furthermore, no cytotoxic effects were observed in both experiments. Therefore, no EC_1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

European and national legislation trigger non-animal testing for skin sensitisation. Using IATA, a weight of evidence approach allows non-animal testing for skin sensitisation potential of a substance registered for REACH registration 2018. Three EURL ECVAM validated methods are recommended for use of integrated strategies rather than as stand-alone tests, to classify substances for skin sensitisation hazard. In order to assess the sensitising potential of the test substance the Direct Peptide Assay (OECD TG 442 C), the KeratinoSensTM (OECD TG 442D) and the human Cell Line Activation Test h-CLAT (OECD TG 442E)) were used. As two out of three in vitro tests with the test substance registered were negative in result, the test substance is predicted to be a non-sensitizer.

Based on the available data and 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 registered has not to be classified.