Registration Dossier

Administrative data

Description of key information

Skin sensitization (OECD 442D): not sensitizing

Skin sensitization (OECD 442C): not sensitizing

Skin sensitization (OECD 442E): sensitizing

Based on the available in vitro tests (DPRA, h-CLAT and KeratinoSens) the test item is not considered to be a skin 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:
July - December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of study:
direct peptide binding assay
Test material information:
Composition 1
Details on study design:
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine-or lysine-containing peptide following 24 hours incubation with the test chemical at 25ºC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteineand lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Used Chemicals:
Synthetic peptide containing Cysteine: Batch 1556171, Purity 95% (by HPLC), Ac-RFAACAA-OH
Synthetic peptide containing Lysine: Batch 1556172, Purtiy 94% (by HPLC), Ac-RFAAKAA-OH
Cinnamic Aldehyde (Positive control): Batch MKBR2427V, Purity 98.7%

Apparatus:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Preparation of Peptide Stock Solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
Preparation of Peptide Calibration Standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.
Preparation of Stability Controls and Precision Controls:
Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile.
Preparation of Positive Control Stock Solution and Test Item Stock Solution:
100 mM stock solutions in acetonitrile of the positive control chemical (Cinnamic Aldehyde) and the test item were prepared.
Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
Acetonitrile solutions of the test item and the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
Acetonitrile solutions of the test item and the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation:
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Positive control results:
Positive control values for cystein: 71,3 %, SD 0.25 %
Positive control values for lysine: 44.6 %, SD 0.87 %
The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be <14.9% for the percent cysteine depletion and <11.6% for the percent lysine depletion. The critera are met.
Key result
Parameter:
other: cystein depletion (%)
Run / experiment:
1
Value:
> -2.26 - < -1.4
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Parameter:
other: Mean lysine depletion (%)
Run / experiment:
1
Value:
> -0.08 - < 0.456
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Other effects / acceptance of results:
All analytical acceptance criteria were met:
Acceptance criteria for Cysteine and Lysine (Standard Linearity): r²>0.99, achieved results: r²>0.999
Acceptance criteria for Cysteine and Lysine respectively (Reference controls): 0.45-0.55 mM (CV <14.9%) and 0.45-0.55 mM (CV <11.6%), achieved results: B: 0.499 mM (CV 0.75%, n=6) and B: 0.504 mM (CV 0.32%, n=6)
Acceptance criteria for Cysteine and Lysine (Test item): SD<14.9% and SD<11.6%, achieved results: SD 0.43% (n=3) and SD 0.30% (n=3)

Reactivity is classed as no to minimal, the DPRA prediction is negative and the test item is therefore a potential non skin sensitizer. A partial co-elution peak was observed in the Cysteine assay, however this is considered to be of minimal impact as the cysteine peak was consistently integrated in all three sample chromatograms. No co-elution peaks occurred in the Lysine assay.

Table 5: Individual Achieved Depletion Values of Cysteine Peptide Depletion

Sample

Peak area (μV.sec)

Peptide concentration(μg/mL) 1

Peptide Depletion (%) 2

Mean Depletion (%)

SD (%)

Positive control

253903

107.35

71.4

71.3

0.25

257128

108.71

71.0

252885

106.91

71.5

Test item

902625

382.01

-1.72

-0.179

0.43

899767

380.80

-1.40

907371

384.02

-2.26

 

SD Standard Deviation

1 Samples prepared at a concentration of 376 μg/mL (0.5 mM)

2 Calculated against a mean Reference Control area of 887340 μV.sec (n=6)

 

 

Table 6: Individual Achieved Depletion Values of Lysine Peptide Depletion

Sample

Peak area (μV.sec)

Peptide concentration (μg/mL) 1

Peptide Depletion (%) 2

Mean Depletion (%)

SD (%)

Positive control

428621

212.34

45.6

44.6*

0.87

439007

217.50

44.3

441606

218.79

43.9

Test item

784117

389.07

0.456

0.112

0.30

788402

391.20

-0.0880

787965

390.98

-0.0320

SD Standard Deviation

1 Samples prepared at a concentration of 388μg/mL (0.5 mM)

2 Calculated against a mean Reference Control area of 787710 μV.sec (n=6)

* Individual and mean results are all outside of the historical data achieved parameters however all results are within acceptance criteria for the assay.

 

Interpretation of results:
other: no skin sensitizing potential based on the key event "protein reactivity"
Conclusions:
In the Direct Peptide Reactivity Assay (DPRA) according to OECD 422C the test item showed no to minimal reactivity and is therefore predicted to be a non-skin sensitizer.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. All analytical acceptance criteria for each peptide run were met. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. There was no reactivity (no depletion) in the presence of either peptide which places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted by DPRA to be a non-skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2017 - January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Qualifier:
according to
Guideline:
other: KeratinoSens, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
July 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Details on study design:
Cell line: KeratinoSens (Human transgenic keratinocyte cell line derived from HaCaT cells)

Material:
Luminometer: TriStar² Multimode reader LB 942 (Berthold)
Spectralphotometer: TriStar² Multimode reader LB 942 (Berthold)
Culture medium 1: D-MEM (Cat. No. 21885-025) + 10 % FBS + 1 % geneticin (Cat. No 10131-027)
Culture medium 2: D-MEM (Cat. No. 21885-025) + 10 % FBS
Culture medium 3: D-MEM (Cat. No. 21885-025) + 1 % FBS
Luciferase Assay System Kit: Promega (Cat. No E1501)
MTT Solution: 5 mg/mL MTT (CAS 298-93-1) in DPBS

Controls:
Negative control (NC): 1% DMSO (v/v) in culture medium 3
Positive control (PC): Cinnamic aldehyde (CA, CAS no. 104-55-2) at 4, 8, 16, 31 and 64 μg/mL with 1% DMSO (v/v)
Blank control: Culture medium 3 without cells

Test substance preparation:
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity >99%; AppliChem; Lot No.: 0000978834). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 (from 0.098 to 200 mM). Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Test concentrations:
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM

Experimental procedure:
A cell suspension of 8 x 104cells/mL in assay medium was prepared. 125 uL of the cell suspension corresponding to 1 x 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C + 1 °C and 5% C02. Thereafter, the assay medium was discarded and replaced by 150 uL test item exposure medium. 50 uL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h + 1 h at 37 °C ± 1 °C and 5% C02.

Luciferase activity
After 48 h + 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 uL test item exposure medium. 27 uL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% C02. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% C02 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at A = 600 nm.

Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.

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 EC15 value of the positive control is within two standard deviations of the historical mean of the test facility
- 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.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled.
Key result
Parameter:
other: luciferase induction
Run / experiment:
1 & 2
Value:
< 1.5
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
The controls confirmed the validity of the study. The calculated EC15 was between 7 and 34 µM (21.59 µM in experiment 1; 8.67 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (6.4% in experiment 1; 10.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled. In the second experiment the threshold was slightly increased with a fold induction of 8.59. As in both experiments the values of the test item were very comparable and all other acceptance criteria were met, this slight increase was accepted and the validity of the study not affected.

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

In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. 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.

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [MM]

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

1.06

1.08

1.12

1.09

0.03

 

8.00

1.11

1.28

1.34

1.24

0.12

 

16.00

1.27

1.31

1.43

1.33

0.08

 

32.00

1.52

1.47

2.43

1.81

0.54

 

64.00

2.17

2.30

2.33

2.27

0.09

*

Test Item

0.98

1.01

1.08

1.12

1.07

0.06

 

1.95

0.93

0.94

1.10

0.99

0.09

 

3.91

1.01

1.24

1.00

1.08

0.13

 

7.81

0.92

0.98

0.90

0.93

0.04

 

15.63

0.99

0.93

0.96

0.96

0.03

 

31.25

0.96

1.03

0.94

0.98

0.05

 

 

62.50

1.01

0.93

0.95

0.97

0.04

 

125.00

1.03

1.08

1.00

1.03

0.04

 

250.00

1.26

1.01

1.13

1.13

0.13

 

500.00

0.97

0.93

1.17

1.03

0.13

 

1000.00

1.00

1.05

1.07

1.04

0.04

 

2000.00

1.10

0.92

1.16

1.06

0.13

 

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

 

Table 3: Induction of Luciferase Activity Experiment 2

 

Experiment 2

Concentration [MM]

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

1.18

1.42

1.01

1.20

0.20

 

8.00

1.37

1.41

1.59

1.46

0.12

 

16.00

1.76

2.08

2.05

1.96

0.18

 

32.00

3.11

3.79

3.43

3.44

0.34

*

64.00

6.71

9.59

9.47

8.59

1.63

*

Test Item

0.98

[4.19]

0.93

1.06

2.06

1.85

 

1.95

0.94

0.83

1.03

0.93

0.10

 

3.91

1.01

0.89

1.13

1.01

0.12

 

7.81

0.86

0.91

1.09

0.95

0.12

 

15.63

0.85

0.79

1.11

0.91

0.17

 

31.25

1.27

0.80

1.01

1.03

0.24

 

62.50

0.65

0.78

0.78

0.74

0.08

 

125.00

0.64

0.63

0.82

0.70

0.11

 

250.00

0.44

0.51

0.79

0.58

0.18

 

500.00

0.34

0.41

0.43

0.40

0.05

 

1000.00

0.29

0.32

0.34

0.32

0.03

 

2000.00

0.45

0.45

0.45

0.45

0.00

 

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

[outlier] according to statistical test of Nalimov; Therefore, the lowest concentration was excluded from further calculations.



Interpretation of results:
other: no skin sensitizing potential based on the key event "activation of keratinocytes"
Conclusions:
In this study according to OECD 442D 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.
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. Based on a molecular weight of 122.17 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 the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. 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. The controls confirmed the validity of the study. The calculated EC15 was between 7 and 34 µM (21.59 µM in experiment 1; 8.67 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (6.4% in experiment 1; 10.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 uM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled. In the second experiment the threshold was slightly increased with a fold induction of 8.59. As in both experiments the values of the test item were very comparable and all other acceptance criteria were met, this slight increase was accepted and the validity of the study not affected.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2017 - Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
Qualifier:
according to
Guideline:
other: Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency.
Version / remarks:
Toxicol In Vitro. 2012 Oct; 26(7):1150-60.
Qualifier:
according to
Guideline:
other: Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay
Version / remarks:
Altern Lab Anim. 2010 Aug; 38(4):275-84.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
activation of dentritic cells
Test material information:
Composition 1
Details on study design:
An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of the test item in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) are determined by XTT tests.
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
Prior to using the cells for testing, the cells were qualified by conducting a reactivity check by using the positive controls 2,4-Dinitrochlorobenzene (DNCB) (CAS no 97-00-7), nickel sulfate (NiSO4) (CASn. 10101-97-0) and the negative control, lactic acid (CAS no 50-21-5), two weeks after thawing. Only the cells which passed the reactivity check were used in the assay. The passage numbers of the used THP-1 cells were 19 and 22 in the XTT assay and 21 and 22 in the h-CLAT for runs 1 and 2, respectively.
Culture Medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.
Preparation and Seeding of THP-1 Cells: For testing, THP-1 cells were seeded at a density of either 0.1 x 106 cell/mL or 0.2 x 106 cells/mL and pre-cultured in culture flasks for about 72 hours or at least 48 hours, respectively. On the day of testing, cells harvested form the culture flask were resuspended with fresh culture medium at 2 x 106 cells/mL. On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 106 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9 - 1 × 106 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.
Dose Finding Assay (XTT Test): The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
Experimental Design and Procedures of h-CLAT: The test item was tested in two independent runs. For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × mean CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
Staining of the Cells: The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
Sample Preparation for Measurement: After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.
Flow Cytometry Acquisition: Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
The following acquisition plots were prepared for acquisition:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH). The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
Acquisition: Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated for each concentration as follows:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)

The cell viability is calculated as follows for each concentration:
Cell viability (%) = (mean cytotox of vehicle control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.

The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the vehicle control should be more than 90% in comparison to the negative control.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability.
- The cell viability of at least 4 doses in each experiment should be ≥ 50%.
Positive control results:
The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value at second concentration of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the respective threshold of 200 %.
Parameter:
other: EC200% (CD54): the concentration resulting in a RFI of 200%
Run / experiment:
1
Value:
> 966
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC200% (CD54): the concentration resulting in a RFI of 200%
Run / experiment:
2
Value:
> 966
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC150% (CD86): the concentration resulting in a RFI of 150%
Run / experiment:
1
Value:
< 323
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC150% (CD86): the concentration resulting in a RFI of 150%
Run / experiment:
2
Value:
> 466
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
The RFI of CD54 was not greater than 200% in any concentration of both independent runs. The RFI of CD86 was greater than 150% in at least one concentration of both independent runs. In addition, a dose response could be observed for both markers in both experiments. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT.

Table 2: Results of the first h-CLAT run for the Test Item

 

Concentration (μg/mL)

Antibody / ISO

MFIGeoMean(FITC)

MFI-ISO

RFI (%)

Cyto(Geo)GeoMean(7-AAD)

Mean Cyto

Viability (%)

Medium control

-

ISO

1.79

 

 

3.30

2.9

100.0

CD54

3.16

1.37

100.0

2.88

CD86

4.40

2.61

100.0

2.55

DMSO control

-

ISO

1.81

 

 

3.19

2.8

100.0

CD54

3.29

1.48

100.0

2.62

CD86

4.25

2.44

100.0

2.44

Positive control (DNCB)

2

ISO

2.11

 

 

4.42

3.4

81.8

CD54

6.13

4.02

271.6

3.31

CD86

15.05

12.94

530.3

2.36

3

ISO

2.11

 

 

4.66

3.5

79.4

CD54

6.97

4.86

328.4

3.37

CD86

16.91

14.80

606.6

2.36

Test Item

323

ISO

1.88

 

 

3.43

2.9

100.2

CD54

3.23

1.35

98.5

2.97

CD86

5.99

4.11

157.5

2.31

388

ISO

1.87

 

 

3.27

2.8

103.7

CD54

3.28

1.41

102.9

2.77

CD86

4.92

3.05

116.9

2.38

466

ISO

1.89

 

 

3.31

2.8

104.8

CD54

3.27

1.38

100.7

2.84

CD86

5.97

4.08

156.3

2.18

559

ISO

1.96

 

 

3.30

2.8

104.4

CD54

3.40

1.44

105.1

2.81

CD86

5.69

3.73

142.9

2.25

671

ISO

2.00

 

 

3.57

3.0

98.0

CD54

3.69

1.69

123.4

3.06

CD86

6.54

4.54

173.9

2.28

805

ISO

2.09

 

 

3.84

3.2

92.0

CD54

3.90

1.81

132.1

3.22

CD86

7.82

5.73

219.5

2.43

966

ISO

2.22

 

 

4.66

3.7

77.9

CD54

4.38

2.16

157.7

3.82

CD86

10.24

8.02

307.3

2.72

1159

ISO

5.47

 

 

67.35

52.4

5.6

CD54

8.46

2.99

218.2

66.77

CD86

141.26

135.79

5202.7

23.12

Test item concentration 1159 μg/mL: cell viability below 50%, data excluded from the evaluation

 

 

Table 3: Results of the second h-CLAT run for the Test Item 

 

Concentration (μg/mL)

Antibody / ISO

MFIGeoMean(FITC)

MFI-ISO

RFI (%)

Cyto(Geo)GeoMean(7-AAD)

Mean Cyto

Viability (%)

Medium control

-

ISO

1.91

 

 

3.98

4.0

100.0

CD54

3.24

1.33

100.0

3.82

CD86

3.53

1.62

100.0

4.11

DMSO control

-

ISO

1.87

 

 

4.25

3.9

100.0

CD54

3.45

1.58

100.0

3.70

CD86

3.20

1.33

100.0

3.88

Positive control (DNCB)

2

ISO

2.13

 

 

6.37

4.3

91.5

CD54

5.18

3.05

193.0#

3.87

CD86

13.33

11.20

842.1

2.69

3

ISO

2.31

 

 

6.77

4.9

80.4

CD54

5.90

3.59

227.2

5.34

CD86

21.82

19.51

1466.9

2.60

Test Item

323

ISO

1.97

 

 

4.34

3.8

104.6

CD54

3.30

1.33

100.0

3.80

CD86

4.19

2.22

137.0

3.25

388

ISO

1.97

 

 

4.16

3.5

112.8

CD54

3.07

1.10

82.7

3.19

CD86

4.06

2.09

126.0

3.21

466

ISO

1.84

 

 

4.26

3.8

104.7

CD54

3.10

1.26

94.7

3.72

CD86

4.18

2.34

144.4

3.40

559

ISO

1.94

 

 

4.34

3.7

106.8

CD54

3.24

1.30

97.7

3.71

CD86

4.99

3.05

188.3

3.10

671

ISO

2.25

 

 

4.12

3.6

111.7

CD54

3.67

1.42

106.8

3.46

CD86

6.12

3.87

238.9

3.08

805

ISO

2.01

 

 

4.66

4.0

99.7

CD54

3.64

1.63

122.6

4.00

CD86

5.50

3.49

215.4

3.28

966

ISO

2.20

 

 

5.88

4.9

81.3

CD54

4.37

2.17

163.2

4.87

CD86

8.40

6.20

382.7

3.90

1159

ISO

2.99

 

 

19.58

15.9

25.0

CD54

5.94

2.95

221.8

16.93

CD86

28.25

25.26

1559.3

11.04

Test item concentration 1159 μg/mL: cell viability below 50%, data excluded from the evaluation

#CD54 RFI value of the positive control (2.0μg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Interpretation of results:
other: skin sensitizing potential based on the key event "activation of dendritic cells"
Conclusions:
In this study according to OECD 442E the test item activated THP-1 cells unter the test conditions in at least two independent experiment runs. Therefore, the test item can be considered positive.
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.
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test item stable suspended in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 μg/mL up to the highest tested concentration (5000 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 966.2 μg/mL. The following concentrations of the test item (stable suspended in culture medium) were tested in the main experiment (h-CLAT): 323, 388, 466, 559, 671, 805, 966 and 1159 μg/mL. The test item with a log Pow of 1.67 was tested in 2 independent runs. The highest concentration of both runs was excluded from the evaluation, since the cell viability was < 50%. The RFI of CD54 was not greater than 200% in any concentration of both independent runs. The RFI of CD86 was greater than 150% in at least one concentration of both independent runs. In addition, a dose response could be observed for both markers in both experiments. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value at second concentration of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the respective threshold of 200 %. In conclusion, the test item with a log Pow of 1.67 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

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

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone in-house validation in the test facility using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of the in-house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90 % and an accuracy of 90 %.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA

LuSens/

KeratinoSensTM

MUSST/

h-CLAT

Test battery evaluation

positive

positive

positive

sensitizer

positive

positive

negative

sensitizer

positive

negative

positive

sensitizer

positive

negative

negative

non-sensitizer

negative

positive

positive

sensitizer

negative

positive

negative

non-sensitizer

negative

negative

positive

non-sensitizer

negative

negative

negative

non-sensitizer

Each individual assay was performed under GLP-conditions and the cell based assays LuSens and MUSST consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays. The DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). An OECD testing guideline for the dentritic cell activation assay is under preparation.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

In accordance with the published evaluation scheme (Bauchet al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is judged not to be a skin sensitizer.

Details on the two assessed tests:

OECD 442C:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. All analytical acceptance criteria for each peptide run were met. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. There was no reactivity (no depletion) in the presence of either peptide which places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted by DPRA to be a non-skin sensitizer.

OECD 442D:

The in vitro KeratinoSens™ assay according to OECD 422D 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 item was dissolved in DMSO. Based on a molecular weight of 122.17 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 uM 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, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. 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. The controls confirmed the validity of the study. The calculated EC15 was between 7 and 34 uM (21.59 uM in experiment 1; 8.67 uM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (6.4% in experiment 1; 10.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 uM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled. In the second experiment the threshold was slightly increased with a fold induction of 8.59. As in both experiments the values of the test item were very comparable and all other acceptance criteria were met, this slight increase was accepted and the validity of the study not affected.

OECD 442E:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test item stable suspended in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 μg/mL up to the highest tested concentration (5000 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 966.2 μg/mL. The following concentrations of the test item (stable suspended in culture medium) were tested in the main experiment (h-CLAT): 323, 388, 466, 559, 671, 805, 966 and 1159 μg/mL. The test item with a log Pow of 1.67 was tested in 2 independent runs. The highest concentration of both runs was excluded from the evaluation, since the cell viability was < 50%. The RFI of CD54 was not greater than 200% in any concentration of both independent runs. The RFI of CD86 was greater than 150% in at least one concentration of both independent runs. In addition, a dose response could be observed for both markers in both experiments. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value at second concentration of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the respective threshold of 200 %. In conclusion, the test item with a log Pow of 1.67 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Overall conclusion

Considering the three studies, the test substance is not considered to be a skin sensitizer.

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. Based on available data on skin sensitization the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.