Registration Dossier

Diss Factsheets

Administrative data

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

Referenceopen allclose all

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%.

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.

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 (Imax) was determined with 1.11 in experiment 1 and 1.15 inexperiment 2. Furthermore, no cytotoxic effects were observed in both experiments. Therefore, no EC1.5 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.