Registration Dossier

Administrative data

Description of key information

DPRA: The test item was negative in the DPRA and was classified in the "no or minimal reactivity class" using the Cysteine 1:10 / Lysine 1:50 prediction model.

KeratinoSens: The test item was negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Additionally, QSAR estimation of the skin sensitising potential of the components of the substance was performed. All components triggered no alerts for skin sensitisation and therefore were predicted as non-sensitisers.

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:
weight of evidence
Study period:
2019-04-16 to 2019-07-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

Synthetic peptides:
Cysteine- (SPCC) containing peptide: Ac-RFAACAA-COOH (MW=750.9 g/mol)
Lysine- (SPCL) containing peptide: Ac-RFAAKAA-COOH (MW=775.9 g/mol)

Controls:
For Cysteine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, SPCC stock solution and ACN
Co-elution control: Phosphate buffer pH 7.5, ACN and the test substance without peptide.
For Lysine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, and SPCL stock solution
Co-elution control: Ammonium acetate buffer pH 10.2 and the test substance without peptide.
Test substance preparation:
22.52 mg and 31.47 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1136 μL and 1587 μL ACN, respectively, to obtain 100 mM solutions. No correc tion for the purity/composition of the test item was performed.

Vehicle: Acetonitrile, ACN
Reason for the vehicle: A solubility test to assesse the test items solubility in different solvents was performed before the main test. ACN was chosed as the best solvent in which the test item dissolved and did not for precipitation or cloudy solution.

Test Item Samples:
- Test item solution, ACN and SPCC stock solution
- Test item and SPCL stock solution

Preparation of peptide stock solutions:
SPCC - A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 11.0 mg of SPCC in 21.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
SPCL - A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.4 mg of SPCL in 20.08 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes. Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.

Experimental procedure:
After preparation, the samples were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation period was 26 hours for RCcysB-samples and 25 hours for the RClysBsamples. All samples were analysed with HPLC analysis. Prior to HPLC analysis the samples were visually inspected for precipitation. The samples that showed precipitation/phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
Key result
Parameter:
other: mean % SPCC depletion
Value:
1.4
Positive controls validity:
valid
Remarks:
80 ± 0.1 %
Key result
Parameter:
other: mean % SPCL depletion
Value:
0.1
Positive controls validity:
valid
Remarks:
58.8 ± 0.7 %
Key result
Parameter:
other: mean % depletion
Value:
0.8
Positive controls validity:
valid

Upon preparation as well as after incubation of the SPCC test item samples a precipitate was observed. Upon preparation of the SPCL test item samples a phase separation was observed while after incubation of these samples a precipitate was observed.

In the cysteine reactivity assay the test item showed 1.4 % SPCC depletion while in the lysine reactivity assay the test item showed 0.1 % SPCL depletion. The mean of the SPCC and SPCL depletion was 0.8 % and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Table 1. SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

 

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

Test item

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

1.4 %

± 0.4 %

0.1 %

± 0.2 %

0.8 %

Negative: No or minimal reactivity

  SD = Standard Deviation

Table 2. SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code

Peak area at 220 nm (μAU)

Concentration (mM)

SPCC Depletion

PCcys-1

417946

0.100

79.9 %

PCcys-2

414846

0.099

80.0 %

PCcys-3

413080

0.099

80.1 %

 

 

Mean

80.0 %

 

 

SD

0.1 %

SD = Standard Deviation

Table 3. SPCC Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test Item Samples

Sample code

Peak area at 220 nm (μAU)

Concentration (mM)

SPCC Depletion

Peak area at 258 nm (μAU)

Area ratio

(A220/A258)

210251/A-cys-1

2056772

0.498

1.0 %

54723

37.59

210251/A-cys-2

2043642

0.495

1.6 %

54528

37.48

210251/A-cys-3

2041676

0.494

1.7 %

54442

37.50

 

 

Mean

1.4 %

NA

37.52

 

 

SD

0.4 %

NA

0.06

SD = Standard Deviation, NA = Not Applicable.

Table 4. SPCL Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code

Peak area at 220 nm (μAU)

Concentration (mM)

SPCC Depletion

PClys-1

666448

0.203

59.5 %

PClys-2

674337

0.206

59.0 %

PClys-3

689184

0.210

58.1 %

 

 

Mean

58.8 %

 

 

SD

0.7 %

SD = Standard Deviation

Table 5. SPCL Peak Area, Concentration, Depletion and Area Ratio (A220/A258) of the Test Item Samples

Sample code

Peak area at 220 nm (μAU)

Concentration (mM)

SPCC Depletion

Peak area at 258 nm (μAU)

Area ratio

(A220/A258)

210251/A-lys-1

1654675

0.507

0.0 %

51344

32.23

210251/A-lys-2

1647770

0.505

0.0 %

53515

30.79

210251/A-lys-3

1647770

0.502

0.3 %

52392

31.28

 

 

Mean

0.1 %

NA

31.43

 

 

SD

0.2 %

NA

0.73

SD = Standard Deviation, NA = Not Applicable.

 

Interpretation of results:
other: negative for dendritic cells activation
Conclusions:
In conclusion, the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and
SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
Executive summary:

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with SPCC or SPCL for 26 and 25 hours, respectively, at 25 °C, the relative peptide concentration was determined by HPLC detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 100 mM and the reference item at 100 mM. As a vehicle acetonitrile (ACN) was used. Synthetic peptide control solutions of SPCC (containing Ac-RFAACAA-COOH) and SPCL (containing Ac-RFAAKAACOOH) were prepared in ACN. Co-eluent samples containing only the test item and the respective buffer were also tested under the same conditions. After the incubation period samples were injected directly into the HPLC system. The concentration of SPCC or SPCL was photometrically determined at 220 and 258 nm. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. Following this model, the mean % SPCC depletion of the test item was 1.4 % and the mean % SPCL depletion was 0.1 %. The average % depletion of the test item was determined to be 0.8 %. The reference control showed mean % SPCC depletion of 80 % and a mean % SPCL depletion of 58.8 %. All validity criteria were met and the test is considered as valid. The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019-03-29 from 2019-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™ (Adopted March, 2018)
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on study design:
- Vehicle: dimethyl sulfoxide (DMSO)
- Solvent/vehicle control (VC): yes, 1 % DMSO in exposure medium. Eighteen wells were tested per plate.
- Positive control (PC): Ethylene dimethacrylate glycol, CAS 50-21-5
- Blank control: on each plate three blank wells were tested (no cells, no treatment).

Test substance preparation:
No correction was made for the composition/purity of the test item. A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation). This concentration was selected as highest concentration for the main assay. In the main experiments a stock solution of 200 mM test item in dimethyl sulfoxide (DMSO) was prepared (colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series in experiment 1 and 1.5-fold dilution series in experiment 2). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM in experiment 1 and 2000, 1333, 889, 593, 395, 263, 176, 117, 78, 52, 35 and 23 μM in experiment 2 (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

Positiv control preparation:
A 2-fold dilution series ranging from 0.78 to 25 mM was prepared in DMSO and was diluted following the same procedure as for the test item to a final concentration of 7.8 to 250 μM (final concentration of DMSO of 1 %). All concentration were tested in triplicates in each assay run.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (e.g. the KeratinoSens™ cell line). Upon receipt, cells were propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
Cell culture:
Basic medium - Dulbecco’s minimal supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum.
Maintenance medium - Dulbecco’s minimal supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum and geneticin (500 μg/mL).
Exposure medium - Dulbecco’s minimal supplemented with 1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum.

Environmental conditions:
All incubations were carried out in a humid atmosphere (43 - 98 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 38.0 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Experimental procedure:
- Subculturing: Cells were subcultured upon reaching 80 - 90 % confluency.
- Plating of Cells: One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. Three plates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1 and P+8 in experiment 2.
- Treatment of Cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for 48 hours at 37 ± 1.0 °C in the presence of 5 % CO2. In total 2 experiments were performed.

Luciferase Activity Measurements:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega are mixed together. The assay plates were removed from the incubator and the medium was removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were then shaken for at least 5 minutes at room temperature. Plates with the cell lysates were then placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time 2 seconds).

Cytotoxicity Assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 - 4 hours at 37 °C in the presence of 5 % CO2. The MTT medium was then removed and cells were lysed overnight by adding 10 % SDS solution to each well. After shaking, the absorption is measured at 570 nm.
Positive control results:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax in experiment 1 was 2.67 and the EC1.5 28 μM and in experiment 2 Imax was was 4,46 and the EC1.5 15 μM, respectively.
Key result
Parameter:
other: Luminescence induction
Run / experiment:
experiment 1 and 2
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: I (max)
Run / experiment:
experiment 1 and 2
Value:
1.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC1.5
Run / experiment:
experiment 1 and 2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Parameter:
other: IC30 in μM
Run / experiment:
experiment 1
Value:
283
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 in μM
Run / experiment:
experiment 2
Value:
136
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 in μM
Run / experiment:
experiment 1
Value:
346
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 in μM
Run / experiment:
experiment 2
Value:
149
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (28 μM and 15 μM in experiment 1 and 2, respectively). A dose-response was observed and the induction at 250 μM was higher than 2-fold (2.67-fold and 4.66-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (11 % and 9.0 % in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) in experiment 1 and 23 – 2000 μM (1.5-fold dilution steps) in experiment 2 for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- The test item showed toxicity. The calculated IC30 was 283 μM and the calculated IC50 was 346 μM.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.67 and the EC1.5 28 μM.

Experiment 2

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- The test item showed toxicity. The calculated IC30 was 136 μM and the calculated IC50 was 149 μM.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.46 and the EC1.5 15 μM.

Table 1. Overview Luminescence Induction and Cell Viability of Cyclodumol Acetate Drum in Experiment 1 and 2

Concentration (μM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp 1 luminescence

1.20

1.07

1.25

1.18

1.34

1.28

1.16

1.24

1.43

0.00

0.00

0.12

Exp 1 viability (%)

105.6

114.0

96.2

97.3

94.2

94.1

99.5

92.7

80.4

1.0

29.3

52.3

Concentration (μM)

23

35

52

78

117

176

263

395

593

889

1333

2000

Exp 2 luminescence

1.31

1.27

1.34

1.43

1.36

0.86

0.00

0.00

0.00

0.01

0.04

0.00

Exp 2 viability (%)

94.8

97.9

101.1

99.3

98.2

8.7

18.5

51.0

-1.7

-3.2

-2.1

-3.7

 

Table 2. Luminescence Induction of Cyclodumol Acetate Drum in Experiment 1

Concentration (μM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Measurement 1

1.16

1.08

1.30

1.22

1.41

1.33

1.17

1.35

1.36

0.00

0.00

0.28

Measurement 2

1.26

1.06

1.27

1.20

1.40

1.31

1.24

1.17

1.43

0.00

0.00

0.08

Measurement 3

1.18

1.06

1.17

1.12

1.22

1.21

1.06

1.19

1.50

0.00

0.00

0.00

Mean (fold-induction)

1.20

1.07

1.25

1.18

1.34

1.28

1.16

1.24

1.43

0.00

0.00

0.12

 

Table 3. Luminescence Induction of Cyclodumol Acetate Drum in Experiment 2

Concentration (μM)

23

35

52

78

117

176

263

395

593

889

1333

2000

Measurement 1

1.71

1.31

1.29

1.43

1.38

0.81

0.00

0.00

0.00

0.00

0.00

0.00

Measurement 2

1.01

1.17

1.34

1.30

1.23

0.62

0.00

0.00

0.00

0.00

0.02

0.00

Measurement 3

1.22

1.32

1.39

1.55

1.47

1.16

0.00

0.00

0.00

0.03

0.11

0.01

Mean (fold-induction)

1.31

1.27

1.34

1.43

1.36

0.86

0.00

0.00

0.00

0.01

0.04

0.00

 

Table 4. Overview EC1.5, Imax, IC30 and IC50 Values

 

EC1.5 (μM)

Imax

IC30 (μM)

IC50 (μM)

Test item Experiment 1

NA

1.43

283

346

Test item Experiment 2

NA

1.43

136

149

Pos Control Experiment 1

28

2.67

NA

NA

Pos Control Experiment 2

15

4.46

NA

NA

NA = Not applicable

Interpretation of results:
other: no skin sensitizing potential based on the key event "activation of keratinocytes"
Conclusions:
The test item showed toxicity (IC30 values of 283 μM and 136 μM and IC50 values of 346 μM and 149 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.43-fold in experiment 1 and 2. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.
Executive summary:

The in vitro KeratinoSens™ assay according to OECD 442D 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.

In the present study the test substance was dissolved in DMSO to a final concentration of 200 mM.

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) in experiment 1 and 23 – 2000 μM (1.5-fold dilution steps) in experiment 2 for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

In experiment 1, no precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 283 μM and the calculated IC50 was 346 μM. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated. The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.67 and the EC1.5 28 μM.

In experiment 2, no precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 136 μM and the calculated IC50 was 149 μM. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated. The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.46 and the EC1.5 15 μM.

Under the condition of this study the test item is, therefore, considered as non-sensitiser. The controls confirmed the validity of the study. The test item showed toxicity (IC30 values of 283 μM and 136 μM and IC50 values of 346 μM and 149 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.43-fold in experiment 1 and 2. In conclusion, the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
An estimation of the skin sensitising properties of the test item using structural alert relationships was perfomed. The results of this estimation are used in a weight of evidence to support experimental in vitro results.
Qualifier:
according to
Guideline:
other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Key result
Parameter:
other: alerts
Value:
0
Remarks on result:
other: Skin sensitisation in mammals is non-sensitiser. No alert matched for QSAR predicted value. The substance contains misclassified features. The substance is within the applicability domain of the model.

Skin sensitisation in mammal is NON-SENSITISER (from KB: Derek KB 2018 1.1)

Overview

Contains misclassified features

Details

Features of the query structure (SMILES: C(COC=O)1CCCCC1) were found in the Lhasa skin sensitisation negative prediction dataset and have been observed in sensitisers that do not match skin sensitisation structural alerts in Derek. The relationship between these features and skin sensitisation may be coincidental or contributory. However, as the query structure does not match any structural alerts or examples for skin sensitisation in Derek, it is predicted to be a non-sensitiser.

Interpretation of results:
other: Derek result: not sensitising
Conclusions:
Using Derek Nexus v6.0.1, the skin sensitising potential of component 1 (1-(3,3-dimethylcyclohexyl)ethyl acetate) was estimated to be non-sensitiser. No alerts were matched. The substance is within the applicability domain of the model.
Executive summary:

The skin sensitising properties were estimated using Derek Nexus v6.0.1. The skin sensitising potential of component 1 (1-(3,3-dimethylcyclohexyl)ethyl acetate) was estimated to be non-sensitiser based on the described QSAR method (Derek, 2018).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
An estimation of the skin sensitising properties of the test item using structural alert relationships was perfomed. The results of this estimation are used in a weight of evidence to support experimental in vitro results.
Qualifier:
according to
Guideline:
other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Key result
Parameter:
other: alerts
Value:
0
Remarks on result:
other: Skin sensitisation in mammals is non-sensitiser. No alert matched for QSAR predicted value. The substance contains misclassified features. The substance is within the applicability domain of the model.

Skin sensitisation in mammal is NON-SENSITISER (from KB: Derek KB 2018 1.1)

Overview

Contains misclassified features

Details

Features of the query structure (SMILES: C(C)1CCC(C)(C)CC1) were found in the Lhasa skin sensitisation negative prediction dataset and have been observed in sensitisers that do not match skin sensitisation structural alerts in Derek. The relationship between these features and skin sensitisation may be coincidental or contributory. However, as the query structure does not match any structural alerts or examples for skin sensitisation in Derek, it is predicted to be a non-sensitiser.

Interpretation of results:
other: Derek result: not sensitising
Conclusions:
Using Derek Nexus v6.0.1, the skin sensitising potential of component 2 ([(1S,2R)-2,6,6-trimethylcycloheptyl]acetate) was estimated to be non-sensitiser. No alerts were matched. The substance is within the applicability domain of the model.
Executive summary:

The skin sensitising properties were estimated using Derek Nexus v6.0.1. The skin sensitising potential of component 2 ([(1S,2R)-2,6,6-trimethylcycloheptyl]acetate) was estimated to be non-sensitiser based on the described QSAR method (Derek, 2018).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
An estimation of the skin sensitising properties of the test item using structural alert relationships was perfomed. The results of this estimation are used in a weight of evidence to support experimental in vitro results.
Qualifier:
according to
Guideline:
other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Key result
Parameter:
other: alerts
Value:
0
Remarks on result:
other: Skin sensitisation in mammals is non-sensitiser. No alert matched for QSAR predicted value. The substance contains misclassified features. The substance is within the applicability domain of the model.

Skin sensitisation in mammal is NON-SENSITISER (from KB: Derek KB 2018 1.1)

Overview

Contains misclassified features

Details

Features of the query structure (SMILES: C(C)1CCC(C)(C)CC1) were found in the Lhasa skin sensitisation negative prediction dataset and have been observed in sensitisers that do not match skin sensitisation structural alerts in Derek. The relationship between these features and skin sensitisation may be coincidental or contributory. However, as the query structure does not match any structural alerts or examples for skin sensitisation in Derek, it is predicted to be a non-sensitiser.

Interpretation of results:
other: Derek result: not sensitising
Conclusions:
Using Derek Nexus v6.0.1, the skin sensitising potential of component 3 ([(1S,2S)-2,6,6-trimethylcycloheptyl]acetate) was estimated to be non-sensitiser. No alerts were matched. The substance is within the applicability domain of the model.
Executive summary:

The skin sensitising properties were estimated using Derek Nexus v6.0.1. The skin sensitising potential of component 3 ( [(1S,2S)-2,6,6-trimethylcycloheptyl]acetate) was estimated to be non-sensitiser based on the described QSAR method (Derek, 2018).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

OECD 442C - DPRA

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with SPCC or SPCL for 26 and 25 hours, respectively, at 25 °C, the relative peptide concentration was determined by HPLC detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 100 mM and the reference item at 100 mM. As a vehicle acetonitrile (ACN) was used. Synthetic peptide control solutions of SPCC (containing Ac-RFAACAA-COOH) and SPCL (containing Ac-RFAAKAACOOH) were prepared in ACN. Co-eluent samples containing only the test item and the respective buffer were also tested under the same conditions. After the incubation period samples were injected directly into the HPLC system. The concentration of SPCC or SPCL was photometrically determined at 220 and 258 nm. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. Following this model, the mean % SPCC depletion of the test item was 1.4 % and the mean % SPCL depletion was 0.1 %. The average % depletion of the test item was determined to be 0.8 %. The reference control showed mean % SPCC depletion of 80 % and a mean % SPCL depletion of 58.8 %. All validity criteria were met and the test is considered as valid. The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

OECD 442D - KeratinoSens

The in vitro KeratinoSens™ assay according to OECD 442D 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.

In the present study the test substance was dissolved in DMSO to a final concentration of 200 mM.

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) in experiment 1 and 23 – 2000 μM (1.5-fold dilution steps) in experiment 2 for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

In experiment 1, no precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 283 μM and the calculated IC50 was 346 μM. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated. The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.67 and the EC1.5 28 μM.

In experiment 2, no precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed toxicity. The calculated IC30 was 136 μM and the calculated IC50 was 149 μM. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.43 and therefore no EC1.5 could be calculated. The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.46 and the EC1.5 15 μM.

Under the condition of this study the test item is, therefore, considered as non-sensitiser. The controls confirmed the validity of the study. The test item showed toxicity (IC30 values of 283 μM and 136 μM and IC50 values of 346 μM and 149 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.43-fold in experiment 1 and 2. In conclusion, the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Additionally, QSAR estimation of the skin sensitising potential of the components of the test item were performed using Derek Nexus v6.0.1. All three components triggered no alerts and thus, were considered as non-sensitisers. However, for all three components a misclassified structure was found. Features of the query structure were found in the Lhasa skin sensitisation negative prediction dataset and have been observed in sensitisers that do not match skin sensitisation structural alerts in Derek. The relationship between these features and skin sensitisation may be coincidental or contributory. However, as the query structure does not match any structural alerts or examples for skin sensitisation in Derek, it is predicted to be a non-sensitiser.

In conclusion, based on the observed experimental results and the prediction outcomes from QSAR the test item is considered to not posses any skin sensitising potential and thus, is classified as non-sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as a skin sensitizer under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.