Registration Dossier

Administrative data

Description of key information

OECD 442C: non-sensitizer

OECD 442D: non-sensitizer

OECD 442E: 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:
2019-06-25 to 2019-07-04
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: Cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
Batch No.: BP 1c (reactor mix)
CAS No.: 2290526-77-9
Compounds: Cyclic N-octanoyl-N-methylglucamine 49.9%
Cyclic N-decanoyl-N-methylglucamine 33.2%
N-octanoyl-N-methylglucamine 3.7%
N-decanoyl-N-methylglucamine 2.4%
N-methylglucamine 0.6%
Sorbitol 1.1%
Octanoic acid 0.1%
Decanoic acid 0.2%
Unidentified components 8.5%
Water 0.3%
Physical State: Highly viscous mass (at 20 °C)
Colour: Brown
Stability: Stable
Storage Conditions: At room temperature
Expiry Date: 28 March 2021
Safety Precautions: The routine hygienic procedures were 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 depletion of both peptides was 64.74%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Value:
0.2
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]

STD1

17.9760

0.5340

16.3790

0.5340

STD2

9.1180

0.2670

8.1840

0.2670

STD3

4.4450

0.1335

4.0750

0.1335

STD4

2.2080

0.0667

2.0370

0.0667

STD5

1.0650

0.0334

1.0270

0.0334

STD6

0.4970

0.0167

0.5160

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

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

5.3190

0.1583

69.09

69.28

0.17

0.25

5.2800

0.1572

69.32

5.2600

0.1566

69.44

Test Item

17.1670

0.5086

0.00

0.00

0.00

n/a

17.2070

0.5098

0.00

17.2300

0.5105

0.00

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

6.2210

0.2030

59.84

60.19

0.32

0.53

6.1260

0.1999

60.45

6.1510

0.2007

60.29

Test Item

15.2630

0.4978

0.00

0.20

0.35

173.21

15.2560

0.4975

0.00

15.1470

0.4940

0.61

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

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 Model

Cysteine PPD

ReactivityClass

DPRA Predictio

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

Categorization of the Test Item

Prediction Model

Prediction Model 1

(Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)

Prediction Model 2

(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

0.10

Minimal Reactivity

negative

0.00

Minimal Reactivity

negative

Positive Control

64.74

High Reactivity

positive

69.28

Moderate Reactivity

positive

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is 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 Cyclic Glucamide C8-C10 was dissolved in dist. water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments.

The molecular weight and purity of the mixture was derived from all components (excluding water and unidentified components). Based on the derived molecular weight of 313 g/mol a 100 mM stock solution was prepared. For the water and the unknown components the test item was corrected with a factor of 1.1. All test item preparations were tested in parallel 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 analysis.

All test item solutions were freshly prepared immediately prior to use.

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 were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

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 were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cdist. water : acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was 6.38% (0.10%). The unidentified components seemed not have influenced the test item, therefore, based on the prediction model 1 the test item can be considered as non-sensitiser.

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 64.74%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-25 to 2019-07-16
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 assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
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: Cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
Batch No.: BP 1c (reactor mix)
CAS No.: 2290526-77-9
Compounds: Cyclic N-octanoyl-N-methylglucamine 49.9%
Cyclic N-decanoyl-N-methylglucamine 33.2%
N-octanoyl-N-methylglucamine 3.7%
N-decanoyl-N-methylglucamine 2.4%
N-methylglucamine 0.6%
Sorbitol 1.1%
Octanoic acid 0.1%
Decanoic acid 0.2%
Unidentified components 8.5%
Water 0.3%
Physical State: Highly viscous mass (at 20 °C)
Colour: Brown
Log KOW: Not applicable
Stability: Stable
Storage Conditions: At room temperature
Expiry Date: 28 March 2021
Safety Precautions: The routine hygienic procedures were 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 (347% experiment 1; 306% experiment 2) and
200% for CD54 (338% experiment 1; 477% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
108
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 410.23 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
113
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 237.40 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
108
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 197.84 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
141
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 284.88 µg/mL
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.

Results of the Cell Batch Activation Test (Batch 6)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

85.4

327

>150

85.6

287

>200

yes

pass

NiSO4

100 µg/mL

86.7

251

>150

86.5

269

>200

yes

pass

LA

1000 µg/mL

96.5

76

150

96.6

77

200

no

pass

Results of the Cell Batch Activation Test (Batch 7)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

86.9

422

>150

85.4

647

>200

yes

pass

NiSO4

100 µg/mL

89.1

254

>150

90.0

454

>200

yes

pass

LA

1000 µg/mL

97.9

53

150

98.0

70

200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

97.40

--

95.50

Solvent Control

DMSO

--

97.60

--

95.50

Cyclic Glucamide

C8-C10

C8

7.81

97.20

7.81

95.40

C7

15.63

97.30

15.63

95.30

C6

31.25

96.50

31.25

95.10

C5

62.50

97.20

62.50

95.30

C4

125.00

96.10

125.00

93.90

C3

250.00

94.20

250.00

87.70

C2

500.00

77.00

500.00

47.40

C1

1000.00

2.80

1000.00

7.70

Calculated CV75 [µg/mL]

509.43

311.03

Mean CV75 [µg/mL]

410.23

SD CV 75 [µg/mL]

140.29

CD54 and CD86 Expression Experiment 1

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.3

96.9

96.9

1429

1058

559

870

499

83

102

256

189

Solvent Control

0.20%

96.8

96.6

96.8

1628

1072

585

1043

487

100

100

278

183

DNCB

4.00

85.3

83.7

82.4

4362

2391

743

3619

1648

347

338

587

322

Cyclic Glucamide C8-C10

492.28

19.1

21.4

20.0

1730

1441

911

819

530

79

109

190

158

410.23

58.7

55.6

55.2

1752

1089

629

1123

460

108

94

279

173

341.86

78.2

76.3

75.8

1549

1127

602

947

525

91

108

257

187

284.88

83.0

82.4

81.9

1303

1186

680

623

506

60

104

192

174

237.40

86.5

86.6

84.5

1416

1110

560

856

550

82

113

253

198

197.84

89.7

89.2

88.8

1593

1034

577

1016

457

97

94

276

179

164.86

91.0

91.7

90.5

1443

1040

566

877

474

84

97

255

184

137.39

93.3

92.0

92.0

1560

1031

544

1016

487

97

100

287

190

CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

97.1

97.0

96.7

1275

870

539

736

331

100

68

237

161

Solvent Control

0.20%

97.3

96.8

96.8

1259

1011

523

736

488

100

100

241

193

DNCB

4.0

82.6

81.2

79.6

2937

3013

687

2250

2326

306

477

428

439

Cyclic Glucamide C8-C10

492.28

27.7

26.1

25.9

1226

1165

695

531

470

72

96

176

168

410.23

56.7

56.7

54.7

1079

1239

632

447

607

61

124

171

196

341.86

67.5

67.5

67.7

1058

1241

619

439

622

60

127

171

200

284.88

79.8

77.9

77.5

1227

1243

557

670

686

91

141

220

223

237.40

84.4

84.6

84.4

1191

1144

562

629

582

85

119

212

204

197.84

88.5

88.2

87.3

1341

1096

543

798

553

108

113

247

202

164.86

91.5

90.8

90.8

1256

1011

516

740

495

101

101

243

196

137.39

93.0

92.7

93.1

1321

948

577

744

371

101

76

229

164

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

96.3

-

96.9

pass

96.7

-

97.3

pass

number of test dosed with viability >50% CD86

≥4

7

pass

7

pass

number of test dosed with viability >50% CD54

≥4

7

pass

7

pass

number of test dosed with viability >50% IgG1

≥4

7

pass

7

pass

RFI of positive control of CD86

≥150

347

pass

306

pass

RFI of positive control of CD54

≥200

338

pass

477

pass

RFI of solvent control of CD86

<150

120

pass

100

pass

RFI of solvent control of CD54

<200

98

pass

147

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

256

pass

237

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

278

pass

241

pass

MFI ratio CD54/IgG1for medium control [%]

>105

189

pass

161

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

183

pass

193

pass

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

97.0

1.3

672

number of test doses with viability >50%

-

-

1786

RFI of positive control of CD86

401.0

146.8

112

RFI of positive control of CD54

576.6

312.0

112

RFI of solvent control of CD86

115.0

15.1

112

RFI of solvent control of CD54

118.8

25.5

112

MFI ratio IgG1/CD86 for medium control [%]

202.4

50.0

112

MFI ratio IgG1/CD86 for DMSO control [%]

221.6

58.5

112

MFI ratio IgG1/CD54 for medium control [%]

141.0

24.7

112

MFI ratio IgG1/CD54 for DMSO control [%]

147.7

25.6

112

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item might 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 Cyclic Glucamide C8-C10 was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 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.

A CV75 of 410.23 ± 140.29 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

492.28, 410.23, 341.86, 284.88, 237.40, 197.84, 164.86, 137.39 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 19.1% (CD86), 21.4% (CD54) and 20.0% (isotype IgG1 control) in the first experiment and to 27.7% (CD86), 26.1% (CD54) and 25.9% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-18 to 2019-08-23
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)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
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: Cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
Batch No.: BP 1c (reactor mix)
CAS No 2290526-77-9
Compounds: Cyclic N-octanoyl-N-methylglucamine 49.9%
Cyclic N-decanoyl-N-methylglucamine 33.2%
N-octanoyl-N-methylglucamine 3.7%
N-decanoyl-N-methylglucamine 2.4%
N-methylglucamine 0.6%
Sorbitol 1.1%
Octanoic acid 0.1%
Decanoic acid 0.2%
Unidentified components 8.5%
Water 0.3%
Physical State: highly viscous mass (at 20 °C)
Colour: brown
Log KOW not specified by the Sponsor
Stability: stable
Storage Conditions: at room temperature
Expiry Date: 28 March 2021
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 (3.38 (experiment 1); 2.50 (experiment 2) and 3.32 (experiment 3)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.75
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 500 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
70.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
337.79
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 500 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
74.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Value:
921.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Run / experiment:
other: 3
Parameter:
other: luciferase activity
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration 0.98 µM
Run / experiment:
other: 3
Parameter:
other: cell viability [%]
Value:
82
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 3
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.

Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Exp. 1

Exp. 2

Exp. 3

Mean

SD

Solvent Control

-

100.0

100.0

100.0

100.0

0.0

Positive Control

4.00

107.0

100.4

99.0

102.1

4.3

8.00

104.0

105.8

110.2

106.7

3.2

16.00

116.8

111.0

111.0

113.0

3.3

32.00

107.4

116.5

111.9

112.0

4.5

64.00

104.4

118.9

108.3

110.5

7.5

Test Item

0.98

111.5

105.6

82.0

99.7

15.6

1.95

87.4

98.7

90.4

92.2

5.8

3.91

95.3

99.7

86.8

93.9

6.6

7.81

98.7

96.7

87.3

94.2

6.1

15.63

101.8

99.0

90.1

96.9

6.1

31.25

93.2

89.2

86.8

89.8

3.2

62.50

123.0

97.0

84.9

101.6

19.5

125.00

93.5

89.7

84.4

89.2

4.6

250.00

87.7

83.2

69.5

80.1

9.5

500.00

70.7

74.5

58.9

68.0

8.1

1000.00

16.9

15.0

14.0

15.3

1.5

2000.00

0.6

0.4

0.5

0.5

0.1

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

1.04

1.22

1.26

1.17

0.12

 

8.00

0.99

1.13

1.15

1.09

0.09

 

16.00

1.31

1.41

1.70

1.48

0.20

 

32.00

1.73

1.60

1.91

1.75

0.16

*

64.00

3.89

2.91

3.34

3.38

0.49

*

Test Item

0.98

0.83

1.06

1.15

1.01

0.17

 

1.95

1.09

0.99

0.97

1.02

0.07

 

3.91

0.93

0.95

0.92

0.93

0.01

 

7.81

0.99

0.94

1.14

1.03

0.11

 

15.63

0.88

0.93

1.09

0.96

0.11

 

31.25

0.96

1.00

1.16

1.04

0.10

 

62.50

1.08

1.14

1.03

1.08

0.06

 

125.00

0.95

1.07

1.16

1.06

0.10

 

250.00

1.45

1.29

1.35

1.37

0.08

 

500.00

1.75

1.74

1.76

1.75

0.01

*

1000.00

2.15

2.38

2.19

2.24

0.13

*

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

1.03

0.99

0.99

1.00

0.02

 

8.00

0.96

1.08

0.99

1.01

0.06

 

16.00

1.23

1.29

1.28

1.27

0.03

 

32.00

1.46

1.64

1.48

1.53

0.10

*

64.00

2.50

2.46

2.54

2.50

0.04

*

Test Item

0.98

0.85

0.98

1.00

0.94

0.08

 

1.95

0.90

0.86

0.87

0.87

0.02

 

3.91

0.94

1.02

0.91

0.96

0.05

 

7.81

0.89

0.90

0.84

0.88

0.03

 

15.63

0.85

0.94

1.18

0.99

0.17

 

31.25

0.88

0.95

0.83

0.88

0.06

 

62.50

1.30

0.99

0.90

1.07

0.21

 

125.00

1.02

1.03

0.87

0.97

0.09

 

250.00

1.26

1.16

1.03

1.15

0.12

 

500.00

1.44

1.50

1.37

1.43

0.07

 

1000.00

1.64

1.48

1.42

1.51

0.11

*

2000.00

0.00

0.00

0.00

0.00

0.00

 

Induction of Luciferase Activity Experiment 3

Experiment 3

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

1.17

1.01

1.14

1.11

0.08

 

8.00

1.30

1.14

1.20

1.21

0.08

 

16.00

1.59

1.30

1.33

1.41

0.16

 

32.00

1.89

1.85

1.78

1.84

0.06

*

64.00

3.14

3.68

3.16

3.32

0.30

*

Test Item

0.98

1.37

1.15

1.26

1.26

0.11

 

1.95

1.20

1.08

1.29

1.19

0.10

 

3.91

1.08

1.11

1.05

1.08

0.03

 

7.81

0.99

0.94

1.08

1.00

0.07

 

15.63

1.07

1.00

1.04

1.04

0.04

 

31.25

0.99

1.02

1.00

1.00

0.01

 

62.50

1.01

0.99

1.06

1.02

0.04

 

125.00

0.99

1.07

1.00

1.02

0.04

 

250.00

1.10

0.95

1.11

1.05

0.09

 

500.00

1.14

1.21

1.23

1.19

0.05

 

1000.00

0.94

0.73

0.66

0.78

0.14

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

Induction of Luciferase Activity – Overall Induction

Overall Induction

Conc. [µM]

Fold Induction

Significance

Exp. 1

Exp. 2

Exp. 3

Mean

SD

 

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.17

1.00

1.11

1.09

0.09

 

8.00

1.09

1.01

1.21

1.10

0.10

 

16.00

1.48

1.27

1.41

1.39

0.11

 

32.00

1.75

1.53

1.84

1.70

0.16

*

64.00

3.38

2.50

3.32

3.07

0.50

*

Test Item

0.98

1.01

0.94

1.26

1.07

0.17

 

1.95

1.02

0.87

1.19

1.03

0.16

 

3.91

0.93

0.96

1.08

0.99

0.08

 

7.81

1.03

0.88

1.00

0.97

0.08

 

15.63

0.96

0.99

1.04

1.00

0.04

 

31.25

1.04

0.88

1.00

0.98

0.08

 

62.50

1.08

1.07

1.02

1.06

0.03

 

125.00

1.06

0.97

1.02

1.02

0.04

 

250.00

1.37

1.15

1.05

1.19

0.16

 

500.00

1.75

1.43

1.19

1.46

0.28

 

1000.00

2.24

1.51

0.78

1.51

0.73

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

Additional Parameters

Parameter

Exp. 1

Exp. 2

Exp. 3

Mean

SD

EC1.5[µM]

337.79

921.83

n.a.

629.81

292.02

Imax

2.24

1.51

1.26

1.67

0.51

IC30[µM]

506.15

537.84

245.63

429.87

160.34

IC50[µM]

692.21

705.87

598.85

665.64

58.25

n.a. = not applicable

Acceptance Criteria

Criterion

Range

Exp. 1

pass/
fail

Exp. 2

pass/

fail

Exp. 3

pass/
fail

CV Solvent Control

< 20%

11.6

pass

8.3

pass

7.2

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

2.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

17.37

pass

30.32

pass

19.35

pass

Induction PC at 64 µM

2.00 < x
< 8.00

3.38

pass

2.50

pass

3.32

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96

Interpretation of results:
GHS criteria not met
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 Cyclic Glucamide C8-C10 was dissolved in DMSO. Based on a calculated molecular weight of 312.89 g/mol a stock solution of 200 mM was prepared. The test item contained 8.5% unidentified components, therefore, it was corrected with a correction factor of 1.1.

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 the first experiment, a max luciferase activity (Imax) induction of 2.24 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 16.9%. The lowest tested concentration with a significant luciferase induction >1.5 (1.75) was found to be 500 µM. The corresponding cell viability was >70% (70.7%).The calculated EC1.5 was <1000 µM (337.79 µM).

In the second experiment, a max luciferase activity (Imax) induction of 1.51 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 15.0%. This was the only concentration with a significant luciferase induction >1.5. The calculated EC1.5 was <1000 µM (921.81 µM).

In the third experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

A dose-response for luciferase activity induction could be observed for the first run but not for the third run. Therefore, under the condition of this study, the test item is 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

No skin sensitisation potential is observed in OECD 442C/D/E studies. Therefore, no classification is warrant for the substance according to CLP.