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Description of key information

Three studies are available to assess the skin sensitising potential of the test substance. The test substance showed no chemical reactivity in the Direct Peptide Reactivity Assay (DPRA), the in vitro Human Cell Line Activation Test (h-CLAT) and an Open Epicutaneous Test (OET) used to evaluate the skin sensitising effect of the test item were negative. The first two studies were recent OECD and GLP compliant studies whereas the OET did not follow an OECD guideline but is considered as to be similar to OECD 406. It is not a GLP study.

The test item is considered as a non skin sensitiser.

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:
15.08.2016 - 19.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom
Type of study:
direct peptide binding assay
Details on study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: acetonitrile

CONTROLS
- Positive control: Cinnamic Aldehyde (100 nM solution in acetonitrile)

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA-OH (MW = 751.5 g/mol)
-- Lysine- (K-) containing peptide: Ac-RFAAKAA-OH (MW = 776 g/mol).
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
- Ratios
- C-containing peptide: 0.5 mM peptide, 5 mM test substance
- K-containing peptide: 0.5 mM peptide, 25 mM test substance

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Incubation: at 25°C for minimum 22 hours incubation

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
- Wavelength: 220 nm (UV)

DATA EVALUATION
- % peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B)

EVALUATION RESULTS
- Mean cysteine and lysine % depletion: =<0 - =<6.38; Class 'No or minimal reactivity'; DPRA prediction: Negative
- Mean cysteine and lysine % depletion: =<6.38 - =<22.62; Class 'Low reactivity'; DPRA prediction: Positive
- Mean cysteine and lysine % depletion: =<22.62 - =<42.47; Class 'Moderate reactivity'; DPRA prediction: Positive
- Mean cysteine and lysine % depletion: =<42.47 - =<100; Class 'High reactivity'; DPRA prediction: Positive
Parameter:
other: Peptide depletion
Run / experiment:
C-containing peptide (mM)
Value:
5.46
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Peptide depletion
Run / experiment:
K-containing peptide (mM)
Value:
0
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Mean peptide depletion
Value:
2.73
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
No co-elution peaks were observed in either the Cysteine or Lysine assays. Small micro droplets were observed in the incubated Lysine peptide sample indicative of potential phase separation. As the test item is present in the Lysine incubation samples in excess relative to the amount of peptide (the concentration of the test item estimated to be present was 15.7 mM) then enough test item is considered to have remained in solution so as to ensure the result is valid and hence there is considered to have been no impact on the data reported.
Interpretation of results:
other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
Conclusions:
The test substance showed no peptide reactivity in the DPRA test under the current conditions.
Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in the Direct Peptide Reactivity Assay (DPRA) in chemico assay according to OECD 442C and GLP.

This method quantifies the remaining concentrations of cysteine- or lysine-containing peptides after 24 hours incubation with the test item at 25 +/-2.5 °C. The percentage of depletion of cysteine- and lysine-peptides is calculated and used in a prediction model to assign the test chemical to one of four reactivity classes and in this way discriminate between sensitisers and non-sensitisers.

The test item was dissolved at a 100 mM concentration in acetonitrile (ACN) and 3 samples of the test item were incubated with a typical concentration of 0.667 mM of cysteine in a pH 7.5 phosphate buffer or 0.667 mM of lysine in a pH 10.2 ammonium acetate buffer. Incubation was done for 22 hours. The remaining non-depleted concentrations were determined by HPLC with gradient elution and UV-detector at 220 nm.

The mean C-peptide depletion, caused by the test substance was determined to be 5.46%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.045%.

Negative depletions are considered to be "zero" for the calculation of the mean peptide depletion. Based on the results and the prediction model, the mean depletion was 2.73% and following the depletion model the reactivity class is 'no to minimal reactivity' and the DPRA prediction is negative.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.07.2016 - 30.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline 442E h-CLAT
Version / remarks:
December 2015
Deviations:
yes
Remarks:
Measurement of CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry. The reactivity check for the qualification of the cells was conducted with the positive and negative controls but without nickel sulfate.
Principles of method if other than guideline:
The described deviations to the OECD draft guideline 442E did not have a negative impact on the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
Type of study:
activation of dendritic cells
Details on study design:
TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: ATCC, #TIB-202 (provided by LGC Standards GmbH, Germany)

TEST-SUBSTANCE PREPARATION
- Concentrations: the maximum concentration of the test item was 2500 μg/mL (culture medium), as tested by a solubility test.
- Stock: 1:2 serial dilutions from 2500 μg/mL
- Vehicle: culture medium
- Reason for vehicle: soluble in water

CONTROLS
- Medium and solvent control: culture medium
- Positive control: DNCB (2,4-dinitrochlorobenzene), final concentration: 2 and 3 μg/mL
- Solvent control for the positive control: DMSO, final concentration 0.2%

MEDIUM
- 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)
- FACS Buffer: PBS with 0.1% (w/v) BSA

EXPERIMENTAL PROCEDURE
- Replicates: 3
- Experiments: 3
- Exposure period: 24 ± 0.5 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 ± 1 hours after exposure

DATA EVALUATION
- The cell viability of the h-CLAT experiment is calculated as follows for each concentration of every chemical:
Relative cell viability: % relative cell viability = (Mean cytotox of solvent control cells / Mean cytotox of the test item treated cells)* 100
Where the Mean cytotox is the mean of GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD 54 and GeoMean(7-AAD) CD 86.
- The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100

EVALUATION RESULTS
The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% and/or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be positive in the h-CLAT.
Otherwise it is considered to be negative in the h-CLAT.
In case of different results in both runs, a third run has to be performed.
If the RFI of CD86 is ≥ 150% at any concentration 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 item is considered to be positive.
Otherwise it is considered to be negative.

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the positive control (DNCB), 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 DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL
in DMSO or the highest soluble concentration will be used as the maximal test concentration instead of CV75-based dose, the data for test item 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%.
Parameter:
other: RFI (CD 54) (%)
Run / experiment:
1
Value:
>= 117.5 - <= 152.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: RFI (CD 86) (%)
Run / experiment:
1
Value:
>= 87.4 - <= 148.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: RFI (CD 54) (%)
Run / experiment:
2
Value:
>= 41.4 - <= 79.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: RFI (CD86) (%)
Run / experiment:
2
Value:
>= 90.6 - <= 152.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: RFI (CD 54) (%)
Run / experiment:
3
Value:
>= 83.8 - <= 151.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: RFI (CD 86) (%)
Run / experiment:
3
Value:
>= 72.3 - <= 90.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance criteria were met

Results of the first h-CLAT run for the Test Item

Sample Conc. (µg/mL) RFI (%) CD54 RFI (%) CD 86 Cell Viability (%)
Medium control   100.0 100.0 100.0
DMSO Control   100.0 100.0 100.0
DNCB 2.0 967.1* 504.0# 87.6
3.0 1230.0* 402.9 70.2
test item 29.3 117.5 87.4 89.4
35.2 122.2 102.9 96.1
42.2 120.6 99.4 95.6
50.6 120.6 104.6 90.6
60.8 128.6 96.0 87.5
72.9 122.2 112.1 94.9
87.5P 134.9 106.3 92.7
105.0P 152.4 148.9 91.3

P precipitation /phase separation (oily droplets), are excluded from the evaluation

* RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

# RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

Results of the second h-CLAT run for the Test Item

Sample Conc. (µg/mL) RFI (%) CD54 RFI (%) CD 86 Cell Viability (%)
Medium control   100.0 100.0 100.0
DMSO Control   100.0 100.0 100.0
DNCB 2.0 596.8* 278.8# 84.9
3.0 616.1* 254.4# 72.0
test item 29.3 41.4 101.8 97.6
35.2 68.7 152.9# 101.9
42.2 55.6 133.5 101.1
50.6 69.7 112.9 92.6
60.8 67.7 123.5 88.1
72.9 63.6 94.7 86.2
87.5P 63.6 90.6 84.4
105.0P 79.8 129.4 82.9

P precipitation /phase separation (oily droplets), are excluded from the evaluation

* RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

# RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

Results of the third h-CLAT run for the Test Item

Sample Conc. (µg/mL) RFI (%) CD54 RFI (%) CD 86 Cell Viability (%)
Medium control   100.0 100.0 100.0
DMSO Control   100.0 100.0 100.0
DNCB 2.0 601.0* 243.8# 100.7
3.0 570.9* 276.2# 96.6
test item 29.3 83.8 79.1 94.7
35.2 129.4 86.9 100.5
42.2 130.9 79.8 102.9
50.6 144.1 90.3 96.8
60.8 120.6 77.9 94.7
72.9 138.2 72.3 96.7
87.5P 151.5 81.3 106.1
105.0P 147.1 88.2 98.7

P precipitation /phase separation (oily droplets), are excluded from the evaluation

* RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

# RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

Interpretation of results:
other: h-CLAT prediction: negative (no activation of dendritic cells) according to OECD 442E
Conclusions:
The test item with a log Pow of 3.01 did not activate THP-1 cells up to a concentration of 72.9 μg/mL (limited by phase separation) under the test conditions of this study and is therefore considered to be negative for the respective key event of the sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The test was performed in accordance to the OECD test guideline 442E and under GLP.

The dose 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 156.25 μg/mL up to the highest tested concentration (2500 μg/mL) in both XTT tests which was limited by the solubility of the test item. Precipitation / phase separation (oily droplets) were observed in the wells of the cells treated with 312.5 up to 2500 μg/mL test item concentration. The mean CV75 value of both XTT tests was calculated as 87.3 μg/mL.

The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 29.3, 35.2, 42.2, 50.6, 60.8, 72.9, 87.5 and 105 μg/mL

The test item with a log Pow of 3.01 was tested in 3 independent runs. Due to different results in the first two runs, a third run was necessary for a final decission. The RFI of CD86 and CD54 was not equal to or greater than 150% and 200%, respectively at any concentration in at least 2 of 3 independent run data. Only one concentration (35.2 μg/mL) of run 2 exceeded the threshold for CD86. Since precipitation / phase separation (oily droplets) was observed in the wells of the cells treated with 87.5 and 105 μg/mL test item concentration in the first and second run as well as with 105 μg/mL test item concentration in the third run, these concentrations were excluded from the evaluation.

Therefore, the test item is considered to be negative in the h-CLAT up to a test item concentration of 72.9 μg/mL.

The cell viability of the cells treated with the highest soluble test item concentration did not fall below 90%. However, the cell viability above the 90% threshold meets the acceptance criterion since the highest soluble test item concentration was tested in this study.

In the DMSO solvent 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%.

In conclusion, the test item with a log Pow of 3.01 did not activate THP-1 cells up to a concentration of 72.9 μg/mL (limited by phase separation) under the test conditions of this study. Therefore the test item is considered to be negative 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:

There are three studies available for the current endpoint.

In the first study the skin sensitisation effect of the test item was assessed in the Direct Peptide Reactivity Assay (DPRA) in chemico assay according to OECD 442C and GLP.

Based on the results and the prediction model, the mean depletion was 2.73% and following the depletion model the reactivity class was 'no to minimal reactivity' and the DPRA prediction was negative. Under the current conditions the test item was considered negative for the respective key event of the skin sensitisation Adverse Outcome Pathway (AOP).

In the second study an in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of the test item dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The test was performed in accordance to the OECD test guideline 442E and under GLP.

The test item with a log Pow of 3.01 did not activate THP-1 cells up to a concentration of 72.9 μg/mL (limited by phase separation) under the test conditions of this study. Therefore the test item is considered to be negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Based on the above it is apparent that in vitro testing towards two out of the three key events yielded a negative result. Hence, the test item is not considered to be a skin sensitizer. Further in vitro or in vivo testing is not needed.

The supporting study is an in vivo study, however, not according to OECD or GLP. Nevertheless, the Open Epicutaneous Test (OET) was used to evaluate the skin sensitizing effect of the test item. The test is similar to the OECD 406 and the maximum user concentration was determined. The result indicated that the test item did not elicit skin sensitisation reactions. This finding is in line with the outcome of the two in vitro studies.

It was concluded that the test item is not sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

Taking into account the results of the available in vitro and in vivo tests, the test item is not considered to be as a skin sensitiser.

The available data on skin 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.