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Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (OECD 442C and 442E): negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
9 - 19 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
yes
Remarks:
no information on vehicle control and reference substances provided
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
TEST SYSTEM
- Supplier: AnaSpec
- Cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 90 - 95%
Expiry date: 5 years

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Supplier: SAFC
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

POSITIVE CONTROL PREPARATION
The positive control was prepared at a concentration of 100 mM.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 2 µL
- Column temperature: 30 °C
Key result
Run / experiment:
other: ≥ 22 h incubation
Parameter:
other: % depletion of cysteine-containing peptide
Remarks:
(mean value of 3 replicates)
Value:
-0.996
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Run / experiment:
other: ≥ 22 h incubation
Parameter:
other: % depletion of lysine-containing peptide
Remarks:
(mean value of 3 replicates)
Value:
11.5
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Run / experiment:
other: ≥ 22 h incubation
Parameter:
other: % overall mean depletion
Value:
5.23
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Table 5. Mean peptide depletion of cysteine-containing peptide.

 

Peak area (µV.sec)

Peptide concentration [µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

Test substance

949469

383

-1.01

-0.996 ± 0.06

948717

383

-0.927

949895

383

-1.05

Positive control

234824

96.3

75.0

74.0 ± 3.77

243906

100

74.1

253197

104

73.1

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

**: Calculated against a mean reference control peak area of 940000 μV.sec (n=6)

Table 5. Mean peptide depletion of lysine-containing peptide.

 

Peak area (µV.sec)

Peptide concentration [µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

Test substance

703216

345

10.8

11.5 ± 0.63

697085

342

11.6

694715

341

11.9

Positive control

341222

167

56.7

55.8 ± 7.06

376020

184

52.3

328441

161

58.4

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

**: Calculated against a mean reference control peak area of 788680 μV.sec (n=6)

Interpretation of results:
other: DPRA prediction: negative (no or minimal reactivity)
Remarks:
according to OECD TG 442C
Conclusions:
Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.
Executive summary:

Solutions of 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. The overall result places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 Mar - 13 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test)
Version / remarks:
December 2015
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
activation of dendritic cells
Details on the study design:
TEST CELL LINE
- Source: American Type Culture Collection
- Passage number: 6 - 7 (XTT test); 14 - 16 (h-CLAT)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- Humidity (%): >90
-CO2 (%): 5.0 ± 0.5

The following information relate to details on XTT
The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable concentration showing 75% cell viability (CV75).

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2 - 0.5%

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 250 µg/mL.
1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL

NUMBER OF REPLICATIONS: 4 samples per dose groups were tested in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared to the solvent control is calculated using the formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])
a) Conc. > 75: max. measured concentration with the % of vehicle control > 75%
b) Conc. < 75: min. measured concentration with the % of vehicle control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %

ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the vehicle control is ≥ 70%

The following information relate to details on h-CLAT
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: DMSO
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: Since a CV75 value could not be determined the highest soluble test substance concentration was multiplied with 1.2 (300 µg/mL)
84, 100, 121, 145, 174, 208, 250 and 300 µg/mL

NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in two independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min

MEASUREMENT
- Device: FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:
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%.
Key result
Run / experiment:
other: 24 h incubation
Parameter:
other: relative fluorescence intensity of CD54 (%)
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 24 h incubation
Parameter:
other: relative fluorescence intensity of CD86 (%)
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were not observed following incubation with the test substance up to the highest soluble concentration (250 µg/mL). The viability of the cells spaced within a range of 103.0 to 131.36% in the first experiment and within a range of 86.48 to 96.36% in the second experiment (threshold of cytotoxicity: < 75%). Due to the lack of cytotoxicity, a CV75 value could not be calculated.
h-CLAT
The cell viability was not negatively affected (viability > 90%) in both independent experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%.

Table 1: Results of the first XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Medium control

-

0.555 ± 0.006

0.233

102.52

Vehicle control

-

0.548 ± 0.023

0.234

100.00

Test substance

1.95

0.576 ± 0.019

0.228

110.95

3.91

0.557 ± 0.008

0.233

103.00

7.81

0.585 ± 0.017

0.228

113.58

15.63

0.587 ± 0.025

0.228

114.19

31.25

0.586 ± 0.005

0.230

113.20

62.5

0.565 ± 0.020

0.227

107.40

125

0.597 ± 0.018

0.229

117.22

250

0.638 ± 0.014

0.226

131.36

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 2: Results of the second XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Medium control

-

0.783 ± 0.016

0.283

96.62

Vehicle control

-

0.794 ± 0.016

0.276

100.00

Test substance

1.95

0.768 ± 0.038

0.280

94.13

3.91

0.737 ± 0.051

0.280

88.21

7.81

0.776 ± 0.044

0.277

96.36

15.63

0.762 ± 0.026

0.274

94.26

31.25

0.779 ± 0.015

0.281

96.21

62.5

0.769 ± 0.046

0.281

94.19

125

0.740 ± 0.039

0.277

89.46

250

0.720 ± 0.010

0.272

86.48

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 3. Results of the first h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI GeoMean (FITC)

MFI - ISO

Csto(Geo) GeoMean (7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Medium control

-

ISO

1.92

-

1.83

1.7

-

100.0

CD54

3.02

1.10

1.62

100.0

CD86

4.41

2.49

1.64

100.0

Vehicle control

-

ISO

1.85

-

2.10

1.8

-

100.0

CD54

2.97

1.12

1.70

100.0

CD86

3.89

2.04

1.46

100.0

Positive control

2

ISO

2.31

-

1.85

1.5

-

113.9

CD54

9.87

7.56

1.44

675.0*

CD86

9.65

7.34

1.33

359.8*

3

ISO

2.32

-

2.44

1.8

-

98.1

CD54

10.73

8.41

1.43

750.9*

CD86

10.24

7.92

1.49

388.2*

Test substance

84

ISO

1.98

-

1.95

1.8

-

99.6

CD54

3.20

1.22

1.75

108.9

CD86

4.00

2.02

1.58

99.0

100

ISO

1.95

-

2.03

1.8

-

96.3

CD54

3.21

1.26

1.85

112.5

CD86

3.72

1.77

1.58

99.0

121

ISO

1.98

-

2.44

1.9

-

92.0

CD54

3.12

1.14

1.71

101.8

CD86

4.21

2.23

1.57

109.3

145

ISO

1.96

-

1.89

1.8

-

98.9

CD54

3.05

1.09

1.83

97.3

CD86

3.79

1.83

1.60

89.7

174

ISO

1.91

-

2.06

1.9

-

94.1

CD54

2.90

0.99

1.79

88.4

CD86

3.35

1.44

1.74

70.6

208

ISO

1.91

-

2.01

1.9

-

94.4

CD54

3.07

1.16

1.99

103.6

CD86

3.45

1.54

1.57

75.5

250

ISO

1.93

-

2.40

2.0

-

89.3

CD54

3.16

1.23

1.90

109.8

CD86

3.35

1.42

1.59

69.6

300

ISO

1.94

-

2.32

1.9

-

92.0

CD54

3.01

1.07

1.59

95.5

CD86

3.79

1.85

1.81

90.7

  *: RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).


 Table 4. Results of the second h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI GeoMean (FITC)

MFI - ISO

Csto(Geo) GeoMean (7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Medium control

-

ISO

1.86

-

1.87

1.7

-

100.0

CD54

2.54

0.68

1.97

100.0

CD86

3.84

1.98

1.72

100.0

Vehicle control

-

ISO

1.81

-

1.84

1.8

-

100.0

CD54

2.55

0.74

1.53

100.0

CD86

3.93

2.12

1.77

100.0

Positive control

2

ISO

2.14

-

2.53

1.5

-

113.9

CD54

5.74

3.60

1.57

486.5*

CD86

9.74

7.60

1.38

358.5*

3

ISO

2.10

-

1.97

1.8

-

98.1

CD54

6.29

4.19

1.53

566.2*

CD86

9.20

7.10

1.28

334.9*

Test substance

84

ISO

1.86

-

2.05

1.8

-

99.6

CD54

2.51

0.65

2.14

87.8

CD86

3.25

1.39

1.80

65.6

100

ISO

2.01

-

1.52

1.8

-

96.3

CD54

2.56

0.55

2.01

74.3

CD86

3.16

1.15

1.35

54.2

121

ISO

1.91

-

1.79

1.9

-

92.0

CD54

2.38

0.47

1.67

63.5

CD86

3.70

1.79

1.71

84.4

145

ISO

1.76

-

1.72

1.8

-

98.9

CD54

2.45

0.69

1.59

93.2

CD86

4.33

2.57

1.59

121.2

174

ISO

1.86

-

1.65

1.9

-

94.1

CD54

2.40

0.54

1.53

73.0

CD86

4.37

2.51

1.55

118.4

208

ISO

1.86

-

1.68

1.9

-

94.4

CD54

2.58

0.72

1.52

97.3

CD86

4.12

2.26

1.60

106.6

250

ISO

1.83

-

2.07

2.0

-

89.3

CD54

2.45

0.62

2.43

83.8

CD86

4.22

2.39

1.57

112.7

300

ISO

1.80

-

1.83

1.9

-

92.0

CD54

2.48

0.68

1.45

91.9

CD86

4.30

2.50

1.51

117.9

*: RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:
other: negative
Remarks:
according to OECD TG 442E
Conclusions:
Under the conditions of the Human Cell Line Activation Test the test substance did not influence the expression of cell surface markers associated with the process of activation of monocytes and dendritic cells.
Executive summary:

The test item with a log Pow range between -0.15 to 1.50 was tested in 2 independent runs. The RFIs of CD86 and CD54 were not equal or greater than 150% and 200%, respectively at any dose in both independent run data. Therefore, the test item is considered to be a nonsensitizer.

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

The skin sensitising potential of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2016). In this study the test substance showed no or minimal peptide reactivity.

In a second study, the skin sensitising potential of the test substance was determined by a Human Cell Line Activation Test according to OECD 442E and in compliance with GLP (2016). Based on the obtained results, the test substance did not influence the expression of cell surface markers associated with the process of activation of monocytes and dendritic cells. Thus, the h-CLAT prediction is considered negative.

Based on a weight of evidence approach considering two negative results in the in vitro test battery according to the adverse outcome pathway defined for skin sensitisation, the test substance is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.