Registration Dossier

Administrative data

Description of key information

DPRA:  QSAR prediction, Software: QSAR Toolbox v4.0.0,

DPRA less than 9% (DPRA 13%) for Cys and Lys depletion. No alerts for protein reactivity identified.

QSAR prediction, Toxtree decision tree approach, no reactivity domain found, suspected non-sensitizer

 

Keratinosens: no indication of skin sensitisation (OECD guideline 442D; GLP; RL1);

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

The calculated EC1.5 was < 1000 μM

 

h-CLAT: no indication of skin sensitisation (OECD guideline 442E; GLP; RL1);

RFI of CD86 - at any tested concentration with cell viability # 50% <150

RFI of CD54 - at any tested concentration with cell viability # 50% <200

Overall conclusions

The WoE allows a decision on the absence of skin sensitisation potential

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-02-07 to 2017-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 04, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™)
- Details on study design:

Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. 1E04 cells/well were seeded in either white (luciferase-measurement) or transparent (MTT-assay) 96-well plates for 24 h ± 1 h in assay medium at 37°C ± 1°C and 5% CO2.
After 24 h the medium was discarded and replaced by 150 µL test item exposure medium. The following concentration range of the test item 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 and for the positive control (Cinnamic aldehyde; CAS 104-55-2; > 98%; Sigma; Lot No.: MKBS2662V) the following concentrations were measured: 4.0, 8.0, 16.0, 32.0 and 64.0 µM. The assay was performed in two independently prepared test repetitions.
All plates were sealed and incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

- Luciferase measurement:
After the incubation period (48 h ± 1 h) the medium was aspirated and the 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. Thereafter, the plates were placed in the plate reader and 50 µL luciferase substrate/well was injected by the injector of the plate reader. Luciferase activity was assessed after 1.000 ms for 2.000 ms for each well. An induction of luminescence > 1.5 – fold

- Cell Viability:
The medium of the cell viability plate was removed and replaced with 200 µL test item exposure medium, 27µL MTT solution was added directly to each individual well. The plates were covered with a sealing tape and incubated for 4 h at 37°C ± 1°C and 5 % CO2. Afterwards the medium was replaced by 200 µL 10% SDS solution per well, the plate was sealed with tape again and incubated at 37°C ± 1°C and 5 % CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at 600 nm.

- Acceptance criteria:
The results of the ARE-Nrf-2 Luciferase assay are considered to be reliable if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a Student's two-tailed t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is < 20% in each repetition.

Controls:
Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Positive Control: Cinnamic aldehyde was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Prediction model:
The test item is considered positive in accordance with UN GHS Category 1 if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 200 μg/mL
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 200 μg/mL is considered as inconclusive.
Positive control results:
The positive control (cinnamic aldehyde) values are considered to be valid because they are within the acceptability range demanded by the OECD test guideline 442d and the determined historical data (see also Table 1).
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: EC1.5
Remarks:
µM
Value:
83.1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: IC30 values < than EC1.5 value
Remarks:
no sign of sensitisation
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: EC1.5
Remarks:
µM
Value:
124.33
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: IC30 value was < the EC 1.5 value
Remarks:
no sign of sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table2: Results of the Cytotoxicity Measurement

Concentration [µM]

Cell Viability [%]

Experiment 1

 

Experiment 2

Mean

SD

Solvent Control

-

100

 

100

100

0.0

Positive Control

4.00

100.0

 

119.9

110.0

14.0

8.00

111.2

 

113.3

112.2

1.5

16.00

107.2

 

133.4

120.3

18.5

32.00

110.8

 

130.1

120.5

13.6

64.00

126.0

 

152.3

139.1

18.6

Test Item

0.98

106.6

 

108.0

107.3

1.0

1.95

116.2

 

126.0

121.1

6.9

3.91

104.1

 

114.7

109.4

7.5

7.81

107.6

 

106.0

106.8

1.2

15.63

58.5

 

85.8

72.2

19.3

31.25

50.2

 

90.4

70.3

28.4

62.50

7.2

 

20.5

13.8

9.4

125.00

6.8

 

4.5

5.7

1.6

250.00

0.1

 

0.0

0.1

0.1

500.00

0.1

 

0.0

0.1

0.1

1000.00

0.1

 

0.0

0.1

0.0

2000.00

0.1

 

0.1

0.1

0.0

Table3: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

Positive Control

4.00

1.10

1.22

1.13

1.15

0.06

8.00

1.31

1.20

1.32

1.28

0.07

16.00

1.76

1.49

1.52

1.59

0.15

*

32.00

2.22

2.07

1.88

2.06

0.17

*

64.00

5.29

5.03

5.60

5.31

0.28

*

Test Item

0.98

1.03

1.02

1.05

1.03

0.02

1.95

1.10

1.13

0.99

1.07

0.07

3.91

0.83

0.90

0.93

0.89

0.05

7.81

0.88

0.94

0.85

0.89

0.05

15.63

0.71

0.66

0.60

0.66

0.05

31.25

0.56

0.50

0.45

0.50

0.05

62.50

0.27

0.51

0.74

0.51

0.23

125.00

0.96

2.72

6.88

3.52

3.04

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

Table3: Induction of Luciferase Activity Experiment 2

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

Positive Control

4.00

1.01

1.11

0.96

1.03

0.08

 

8.00

1.31

1.47

1.14

1.31

0.16

 

16.00

1.40

1.28

1.37

1.35

0.07

 

32.00

2.26

2.00

1.89

2.05

0.19

*

64.00

3.36

3.05

3.85

3.42

0.40

*

Test Item

0.98

0.70

0.89

0.86

0.82

0.10

 

1.95

0.66

0.80

0.78

0.75

0.08

 

3.91

0.72

0.91

0.85

0.83

0.10

 

7.81

0.64

1.07

0.83

0.85

0.21

 

15.63

0.39

0.70

0.74

0.61

0.19

 

31.25

0.34

0.64

0.64

0.54

0.18

 

62.50

0.33

0.65

0.65

0.54

0.18

 

125.00

1.72

1.45

1.36

1.51

0.19

*

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

 

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

Table4: Induction of Luciferase Activity – Overall Induction

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.15

1.03

1.09

0.09

 

8.00

1.28

1.31

1.29

0.02

 

16.00

1.59

1.35

1.47

0.17

 

32.00

2.06

2.05

2.06

0.00

*

64.00

5.31

3.42

4.36

1.33

 

Test Item

0.98

1.03

0.82

0.92

0.15

 

1.95

1.07

0.75

0.91

0.23

 

3.91

0.89

0.83

0.86

0.04

 

7.81

0.89

0.85

0.87

0.03

 

15.63

0.66

0.61

0.64

0.03

 

31.25

0.50

0.54

0.52

0.03

 

62.50

0.51

0.54

0.52

0.03

 

125.00

3.52

1.51

2.52

1.42

 

250.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

 

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

Table5: Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control [%]

< 20%

8.1

pass

15.8

pass

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

≥ 1

3.0

pass

2.0

pass

EC1.5 PC [µM]

7 < x < 30 µM

13.66

pass

19.42

pass

Induction PC at 64 µM

2 .00 < x < 8.00

5.31

pass

3.42

pass

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
Executive summary:

In this study conducted according to OECD test guideline 442d (adopted February 04, 2015) transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with D-Glucose reaction products with alcohols C16 -C18 at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37°C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured with a plate reader for 2.000 ms. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide) assay method. The KeratinoSens assay is considered to provide positive results if the following conditions are all met in two of two independent experimental repetitions:

- the  I max   is  higher  than  (>)  1.5  fold  and  statistically  significant  different  as  compared  to  the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);

- the  cellular  viability  is  higher  than  (>)  70%  at  the  lowest  concentration  with  induction  of luciferase activity above 1.5 fold (i.e. at the EC 1.5  determining concentration);

- the EC 1.5  value is less than (<) 1000 µM (or < 200  µg/mL for test chemicals with no defined MW);

- there  is  an  apparent  overall  dose-response  for  luciferase  induction.

In the first experiment, a max luciferase activity (Imax) induction of 3.52 was determined at a test item concentration of 125 μM. The corresponding cell viability was 6.8%. There was no concentration with a significant luciferase induction >1.5. The calculated EC1.5 was < 1000 μM (83.10 μM).

In the second experiment, a max luciferase activity (Imax) induction of 1.51 was determined at a test item concentration of 125 μM. The corresponding cell viability was 4.5%. This was the only concentration with a significant induction >1.5. The calculated EC1.5 was < 1000 μM (124.33 μM).

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

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 (5.31 experiment 1; 3.42 experiment 2). The calculated EC1.5 was between 7 and 30 μM (13.66 experiment 1; 19.42 experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (8.1% experiment 1; 15.8% experiment 2).

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

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-05-17 to 2017-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
Version / remarks:
2015
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was freshly prepared immediately prior to use.

- Solvent Finding
All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mg/mL.

- Final dilution of a dissolved solid, stock liquid or gel:
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%, Honeywell; Lot No.: SZBG1310V, SZBG2930H) at a concentration of 500 mg/mL, respectively. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.

The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium.

The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:
Experimental Procedure

Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 μL FACS buffer. 200 μL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 μg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration according to the equation given in the OECD guidance.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation utilizing the equation given in the OECD guidance. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was solved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 • CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86 (BD, art. no. 555657, Lot No.: 6036848), anti-CD54 (BioLegend, art. no.: 353108, Lot No.: B214036) or mouse IgG1 (isotype, BioLegend, art. no.: 400110, Lot No.: B206037) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.

Controls
1. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
2. Positive Control: 4 μg/mL DNCB

Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO) even if the cell viability is >90%.

Acceptance criteria
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.
Positive control results:
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (196% experiment 1; 406% experiment 2) and 200% for CD54 (253% experiment 1; 359% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: RFI of CD86
Remarks:
at any tested concentration with cell viability ≥ 50%
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: RFI of CD54
Remarks:
at any tested concentration with cell viability ≥ 50%
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Reactivity Check of the Cell Stock: Doubling time of the cells was monitored and found to be 43.9 h which is within the doubling time range specified by the manufacturer (35 - 50 h).
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.
The cell batch was accepted for further testing.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, cell viability of the solvent controls was >90% in both experiments (Exp. 1: 96.3 - 96.6; Exp. 2: 98.0 - 98.2), The the RFI values of the solvent control was not ≥150% for CD86 (Exp. 1: 108; Exp. 2: 112) and not ≥200% for CD54 (Exp. 1: 98; Exp. 2: 111)
- Acceptance criteria met for positive control: yes, the RFI values of the positive control (DNCB) is ≥150% for CD86 (Exp. 1: 196; Exp. 2: 406) and ≥200% for CD54 (Exp. 1: 253; Exp. 2: 359) at a cell viability of >50%
- Acceptance criteria met for variability between replicate measurements: not applicable
- Range of historical values if different from the ones specified in the test guideline: see table

- test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested
- the cell viability was more than 50% in at least four tested concentrations in each run

 Table 1: Results of the Cell Batch Activation Test

 

 

Concentration [μg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

4 µg/mL

91.6

376

91.4

476

Yes

NiSO4

100 µg/mL

89.8

239

89.9

506

Yes

Lactic Acid

1000 µg/mL

96.9

75

97.1

80

No

 

 

Table 2: Results of the Dose Finding Assay

 

 

Experiment 1

 

Experiment 2

 

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

0.00

96.80

0.00

97.30

DMSO Control 

0.00

97.40

0.00

97.30

test item

7.81

96.70

7.81

96.80

15.63

96.20

15.63

96.60

31.25

95.50

31.25

95.30

62.50

89.60

62.50

88.70

125.00

54.90

125.00

62.10

250.00

4.00

250.00

9.00

500.00

4.60

500.00

2.60

1000.00

12.80

1000.00

7.80

Calculated CV75 [µg/mL]

83.66

89.31

Mean CV75 [µg/mL]

86.49

SD CV 75 [µg/mL]

4.00

 

The mean CV75 was derived from two single runs and was found to be 86.5 ± 4.0 μg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 103.8 – 29.0 μg/mL (51.9 – 14.5 mg/mL stock solution)

 

Table 3: CD54 and CD86 Expression Experiment 1

Sample

Conc. [μg/mL]

Cell Viability [%] 

Mean Fluorescence Intensity 

corrected Mean Fluorescence Intensity 

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

 

96.3

96.6

96.5

1591 

989 

470 

1121

519

93 

102 

339

210

DMSO Control 

0.20%

96.5

96.4

96.3

1638 

941 

432 

1206

509

100 

100 

379

218

DNCB

4.0

77.4

79.0

78.6

2892 

1815 

526 

2366

1289

196 

253 

550

345

test item

103.79

8.3

7.6

7.8

1899 

1721 

776 

1123

945

93 

186 

245

222

86.49

58.4

55.5

56.8

1260 

743 

441 

819

302

68 

59 

286

168

72.08

72.7

75.0

76.1

1259 

889 

442 

817

447

68 

88 

285

201

60.06

85.6

87.0

88.4

1380 

1078 

493 

887

585

74 

115 

280

219

50.05

89.8

89.5

88.7

1285 

1040 

489 

796

551

66 

108 

263

213

41.71

92.8

92.0

92.4

1224 

990 

511 

713

479

59 

94 

240

194

34.76

95.0

95.4

94.5

1297 

943 

481 

816

462

68 

91 

270

196

28.97

94.5

94.8

95.2

1428 

944 

467 

961

477

80 

94 

306

202

 

 

Table 4: CD54 and CD86 Expression Experiment 2

Sample

Conc. [μg/mL]

Cell Viability [%] 

Mean Fluorescence Intensity 

corrected Mean Fluorescence Intensity 

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

 

98.1

98.0

98.2

917 

764 

423 

494

341

89 

90 

217

181

DMSO Control 

0.20%

98.2

98.1

98.1

986 

811 

432 

554

379

100 

100 

228

188

DNCB

4.0

87.5

87.6

87.8

2707 

1816 

457 

2250

1359

406 

359 

592

397

test item

103.79

20.2

15.9

14.7

1109 

1190 

626 

483

564

87 

149 

177

190

86.49

66.5

68.4

59.7

1015 

865 

484 

531

381

96 

101 

210

179

72.08

79.5

78.8

71.6

1013 

893 

420 

593

473

107 

125 

241

213

60.06

89.6

90.5

88.2

1177 

1138 

442 

735

696

133 

184 

266

257

50.05

93.7

92.4

91.7

1088 

1074 

459 

629

615

114 

162 

237

234

41.71

93.7

93.1

92.2

1128 

1028 

462 

666

566

120 

149 

244

223

34.76

95.3

94.4

94.7

1185 

984 

455 

730

529

132 

140 

260

216

28.97

95.7

95.6

95.2

1189 

945 

441 

748

504

135 

133 

270

214

 

 

Table 5: Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

96.3 - 96.6

pass

98.0 - 98.2

pass

number of test dosed with viability >50% CD86

≥4

7

pass

7

pass

number of test dosed with viability >50% CD54

≥4

7

pass

7

pass

number of test dosed with viability >50% IgG1

≥4

7

pass

7

pass

RFI of positive control of CD86

≥150

196

pass

406

pass

RFI of positive control of CD54

≥200

253

pass

359

pass

RFI of solvent control of CD86

<150

108

pass

112

pass

RFI of solvent control of CD54

<200

98

pass

111

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

339

pass

217

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

379

pass

228

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

210

pass

181

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

218

pass

188

pass

 

 

Table 6: Historical Data

 

Mean

SD

N

cell viability solvent controls [%]

97.4

1.2

462

number of test doses with viability >50%

-

-

1060

RFI of positive control of CD86

417.5

170.7

77

RFI of positive control of CD54

660.0

319.7

77

RFI of solvent control of CD86

116.9

14.6

77

RFI of solvent control of CD54

124.6

26.9

77

MFI ratio IgG1/CD86 for medium control [%]

193.3

48.6

77

MFI ratio IgG1/CD86 for DMSO control [%]

213.5

60.5

77

MFI ratio IgG1/CD54 for medium control [%]

130.1

16.6

77

 

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser.
Executive summary:

In this study conducted according to OECD test guideline 442E (adopted 29 July 2016) THP-1 cells were incubated with D-Glucose reaction products with alcohols C16 -C18 dissolved in DMSO.

Based on the CV75, the main experiment was performed covering the following concentration steps: 103.8, 86.5, 72.1, 60.1, 50.1, 41.7, 34.8, 29.0 μg/mL

 

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

Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 8.3% (CD86), 7.6% (CD54) and 7.8% (isotype IgG1 control) in the first experiment and to 20.2% (CD86), 15.9% (CD54) and 14.7% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (196% experiment 1; 406% experiment 2) and 200% for CD54 (253% experiment 1; 359% experiment 2) were clearly exceeded.

 

Therefore, the test item is considered to be no skin sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Endpoint:
skin sensitisation: in chemico
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint:
skin sensitisation, other
Remarks:
in silico prediction
Type of information:
(Q)SAR
Adequacy of study:
supporting study
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:
1. SOFTWARE
Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach) version 2.6.13

2. MODEL (incl. version number)
Toxtree v2.6.13

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES = CCCCCCCCCCCCCCCCOC1OC(CO)C(O)C(O)C1O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: skin sensitisation
- Unambiguous algorithm: : identification of structural alerts for aromatic nucleophilic substitution (SNAr), Schiff base formation (SB), Michael-type addition (MA), aliphatic nucleophilic substitution (SN2) and acylation (Ac) (potential mechanisms of action regarded as important for determining a chemical’s skin sensitising potential)

The SMARTS rules aim to identify potential electrophilic fragments and therefore identify a potential hazard associated with a compound, it is up to the user to apply issues relating to bioavailability etc.
The algorithm then attempts to match each of the input SMILES strings against each of the SMARTS patterns. If a chemical matches one or more of the SMARTS patterns within each mechanism then it is assigned to that mechanism. If a chemical matches multiple SMARTS patterns from more than a single mechanism then it is assigned to each of the mechanisms which it matches. A chemical is only assigned to the category ‘non-sensitiser’ if it fails to match any of the SMARTS patterns in any of the mechanistic categories.

Reference
Enoch SJ, Madden JC, Cronin MT, Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach, SAR QSAR Environ Res. 2008;19(5-6):555-78


5. APPLICABILITY DOMAIN
The training set contained a recently published 210 chemical LLNA assay database to derive a set of SMARTS patterns capable of identifying potential mechanisms of action. Having defined the initial SMARTS patterns, a second recently published series of 44 LLNA assay results with mechanisms of action assigned to them was used to validate the rules.

6. ADEQUACY OF THE RESULT
The Toxtree estimation revealed that there are no structural fragments associated with skin sensitisation reactivity domains, thus, based on structure the substance is
considered not to be a sensitizer.
Qualifier:
no guideline followed
Principles of method if other than guideline:
QSAR prediction with Toxtree decision tree approach.
GLP compliance:
no

Results

No skin sensitisation reactivity domains alerts identified. = YES

Alert for SN2 identified. = NO

Alert for Michael Acceptor identified. = NO

Alert for Acyl Transfer agent identified. = NO

Alert for SNAr Identified. = NO

Alert for Schiff base formation identified. = NO

 

No skin sensitisation reactivity domains alerts identified.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on a reliable prediction performed with the Toxtree software, D-Glucose reaction products with alcohols C16 -C18 is not considered to be a skin sensitizer.
Executive summary:

The sensitising potential of D-Glucose reaction products with alcohols C16 -C18 was estimated using the Toxtree decision tree

approach. This QSAR software predicts the sensitising potential via skin sensitisation reactivity domains.

No structural alert was found for D-Glucose reaction products with alcohols C16 -C18. The test item is therefore considered to be

a non-sensitizer.

Endpoint:
skin sensitisation, other
Remarks:
in silico prediction
Type of information:
(Q)SAR
Adequacy of study:
supporting study
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:
1. SOFTWARE
QSAR Toolbox v4.0.0

2. MODEL (incl. version number)
QSAR Toolbox v4.0.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES = CCCCCCCCCCCCCCCCOC1OC(CO)C(O)C(O)C1O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: skin sensitisation
- Unambiguous algorithm: protein binding potency Cys + Lys in DPRA assay
- classification into the following categories:
DPRA above 21% (DPRA 13%)
DPRA less than 9% (DPRA 13%)
Grey zone 9-21% (DPRA 13%)


5. APPLICABILITY DOMAIN
The profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from Direct Peptide Reactivity Assay (DPRA). The DPRA is a reactivity assay which evaluates the ability of chemicals to react with proteins. As model peptides are used reduced glutathione and two synthetic peptides – lysine and cysteine. The reaction time for both lysine and cysteine is 24 hours. The peptide reactivity is reported as percent peptide depletion. The profile contains 73 structural alerts extracted from about 228 chemicals with experimentally measured lysine depletion values. The set of 73 structural alerts are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%). Classification of potency categories is based on analysis published in a collaboration with L`Oreal (Dimitrov et al., 2016).
The data used to derive the structural boundaries have been taken by the following literature sources:

Natsch et al., 2015; Urbisch et al., 2015 and Jaworska et al., 2015 Gerberick, G.F., Vassallo, J.D., Bailey, R.E., Chaney, J.G., Morrall, S.W. and Lepoittevin, J.P. 2004. Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81: 332-343.

Natsch, A. and Gfeller, H. 2008. LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitisation potential. Toxicol. Sci. 106: 464-478.

Natsch, A., Emter, R., Gfeller, H., Haupt, T. and Ellis, G. 2015. Predicting skin sensitizer potency based on in vitro data from KeratinoSens and kinetic peptide binding: global versus domain-based assessment. Toxicol. Sci. 143(2), 319-332.

Jaworska, J., Natsch, A., Ryan, C., Strickland, J., Ashikaga, T., Miyazawa, M. 2015. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch Toxicol, 2355-2383.

Urbisch, D., Mehling, A., Guth, K., Ramirez, T., Honarvar, N., Kolle, S., Landsiedel, R., Jaworska, J., Kern, P., Gerberick, F., Natsch, A., Emter, R., Ashikaga, T., Miyazawa, M., Sakaguchi, H. 2015. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regulatory Toxicology and Pharmacology 71: 337-351.
S. Dimitrov, A. Detroyer, C. Piroird, C. Gomes, J. Eilstein, T. Pauloin, C. Kuseva, H. Ivanova, I. Popova, Y. karakolev, S. Ringeisses, O. Mekenyan, Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example. J Appl Toxicol, 2016, DOI 10.1002/jat.3318


6. ADEQUACY OF THE RESULT
Qualifier:
no guideline followed
Principles of method if other than guideline:
QSAR prediction with QSAR Toolbox v4.0.0
GLP compliance:
no

The substance is classified as: DPRA less than 9% (DPRA 13%) for Cys and Lys depletion. No alerts for protein reactivity were identified.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on a reliable prediction performed with the QSAR toolbox software, D-Glucose reaction products with alcohols C16 -C18 is not considered to be a skin sensitizer.
Executive summary:

The sensitising potential of D-Glucose reaction products with alcohols C16 -C18 was estimated using the QSAR toolbox

approach.

The substance is classified as: DPRA less than 9% (DPRA 13%) for Cys and Lys depletion. No alerts for protein reactivity were identified.

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

It was not feasible to obtain reliable results from a DPRA test (OECD 442C) as, firstly, the substance is a UVCB and, secondarily, has a very low water solubility (0.142 µg/L). Therefore the first molecular key event of the adverse outcome pathway (AOP) was adressed by QSAR prediction (Software: QSAR Toolbox v4.0.0). No alerts for protein reactivity (DPRA less than 9% (DPRA 13%) for Cys and Lys depletion) were identified for D-Glucose, reaction products with alcohols C16-18 (even numbered) by QSAR prediction.

 

The second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, was adressed by an in-vitro KeratinoSens assay (OECD 442D).

In the first experiment, a max luciferase activity (Imax) induction of 3.52 was determined at a test item concentration of 125 μM. The corresponding cell viability was 6.8%. There was no concentration with a significant luciferase induction >1.5. The calculated EC1.5 was < 1000 μM (83.10 μM). In the second experiment, a max luciferase activity (Imax) induction of 1.51 was determined at a test item concentration of 125 μM. The corresponding cell viability was 4.5%. This was the only concentration with a significant induction >1.5. The calculated EC1.5 was < 1000 μM (124.33 μM).

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 was therefore considered as non-sensitiser.

The third molecular key event of the adverse outcome pathway (AOP), events in dendritic cells was adressed by a h-CLAT study conducted according to OECD test guideline 442E (adopted 29 July 2016).

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

Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 8.3% (CD86), 7.6% (CD54) and 7.8% (isotype IgG1 control) in the first experiment and to 20.2% (CD86), 15.9% (CD54) and 14.7% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (196% experiment 1; 406% experiment 2) and 200% for CD54 (253% experiment 1; 359% experiment 2) were clearly exceeded.

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser.


Data integration in a Weight of Evidence Analysis

 

Reference

Study result and/or positive (+ve)/negative (-ve) evidence obtained

Data Reliability

 

 

 

 

Dermal penetration

OECD TG 428, human skin

0.01 %

1

Degradation/Metabolism information

Expert statement

cleavage products, sugar and fatty alcohol do not show alerts for skin sensitisation

 

Non-testing approaches

 

 

 

Protein Binding alerts such as those in the OECD Toolbox, Toxtree

OECD Toolbox
Toxtree

Negative
Negative

2

Testing approaches

 

 

 

Events in keratinocytes

OECD 442 D

negative

1

Events in dendritic cells

OECD 442 E

negative

1

Other relevant information e.g. skin irritation/corrosion data, dermal acute study information (LD50), Ames, in vitro chrom abs

OECD 439

OECD 471, OECD 490, OECD 473

Negative for skin irritation

Negative for genetictoxicity

1

Overall conclusions

The WoE allows a decision on the absence of skin sensitisation potential

 

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on relevant, reliable and adequate data, D-Glucose, reaction products with alcohols C16-18 (even numbered) does not need to be classified and labelled according to Regulation (EC) No 1272/2008 with respect to skin sensitisation.