Registration Dossier

Administrative data

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA test, OECD TG 442C) and does activate keratinocytes ( ARE-Nrf2 luciferase test, OECD TG 422D). Therefore, the substance is predicted to be a skin sensitizer (reference 7.4.1 -1 and -2).

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:
24 June 2019 - 11 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system):
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C.

Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer

Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 156.06 mg test item was dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.

Positive control, reference controls and co-elution control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a co-elution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analysed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.

Data evaluation
HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) started 22 to 26 hours after the test item had been mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours. The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) 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.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Prediction model:
The mean percent cysteine and percent lysine depletion value was calculated for each test item. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an Integrated Approach to Testing and Assessment (IATA). Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.
Positive control results:
Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.
Key result
Parameter:
other: mean peptide depletion in %
Run / experiment:
1st experiment
Value:
8.85
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- No precipitate in the reaction mixture at the end of the incubation time and no co-elution were observed.

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

TABLE 1   Mean of cy steine and lysine % depletion

Test item Art. 851009

Cysteine peptide

Sample

no.

Peak area

Actual
peptide
[mM]

Peak area
reference
control C

Peptide
depletion
[%]

Corrected
peptide
depletion
[%]

Replicate 1
Replicate 2
Replicate 3

45.5235
45.6717
45 7667

0.471
0.473
0.474

49.0051
48.9476
48 6365

6.83
6.53
6.29

6.83
6.53
6.29

Mean
SD
CV

45.661
0.133
0.003

0.473
0.001
0.003

48.8631
0.198
0.004

6.55

0.272

0.042

6.55
0.272
0.042

Lysine peptide

Sample

no.

Peak area

Actual
peptide
[mM]

Peak area
reference
control C

Peptide
depletion
[%]

Corrected
peptide
depletion

[%]

Replicate 1
Replicate 2
Replicate 3

35.2319
35.0659
35.0171

0.437
0.435
0.435

39.4984
39.4912
39.5254

10.82
11.24
11.36

10.82
11.24
11.36

Mean
SD
CV

35.105
0.113
0.003

0.436
0.001
0.003

39.5050

0.0180

0.0005

11.14
0.285
0.026

11.14

0.285
0.026

Acceptance criteria:

SD % depletion Cysteine < 14.9 met:
SD % depletion Lysine < 11.6 met:

YES
YES

Result:

Art. 851009

 

 

 

Mean
depletion
(Cysteine + Lysine)
8.85

Reactivity
Class
(Cysteine + Lysine)
LOW

Reactivity
Class
(Cysteine only)
NEGATIVE

SD = standard deviation
CV = coefficient of variation

TABLE 1  Mean of cysteine and lysine % depletion (continued)

Positive control: Cinnamic aldehyde, vehicle: acetonitrile

Cysteine peptide

 

 

 

 

 

 

 

 

 

Sample

Peak area

Actual

Peak area

Peptide

Corrected

no.

 

peptide

reference

depletion

peptide

 

 

[mM]

control C

[%]

depletion
[%]

Replicate 1

14.9565

0.154

49.0051

69.39

69.39

Replicate 2

14.5266

0.150

48.9476

70.27

70.27

Replicate 3

14.7173

0.152

48.6365

69.88

69.88

Mean

14.733

0.152

48.8631

69.85

69.85

SD

0.215

0.002

0.1983

0.441

0.441

CV

0.015

0.015

0.0041

0.006

0.006

Lysine peptide

Sample

Peak area

Actual

Peak area

Peptide

Corrected

no.

 

peptide

reference

depletion

peptide

 

 

[mM]

control C

[%]

depletion
[%]

Replicate 1

16.7054

0.206

39.4984

57.71

57.71

Replicate 2

17.1234

0.212

39.4912

56.66

56.66

Replicate 3

17.3847

0.215

39.5254

55.99

55.99

Mean

17.071

0.211

39.5050

56.79

56.79

SD

0.343

0.004

0.0180

0.867

0.867

CV

0.020

0.020

0.0005

0.015

0.015

Acceptance criteria:

60.8 < Mean % depletion Cysteine < 100

YES

 

 

40.2< Mean % depletion Lysine < 69.0

YES

 

 

SD % depletion Cysteine < 14.9

 

YES

 

 

SD % depletion

Lysine < 11.6

 

YES

SD = standard deviation
CV = coefficient of variation

 

Interpretation of results:
other: peptide depletion positive
Conclusions:
In this study under the given conditions the test item is considered positive regarding peptide depletion and predicted to be a sensitiser (low reactivity). The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.

Test item-treated samples revealed a cysteine peptide depletion of 6.55% and a lysine peptide depletion of 11.14% (mean peptide depletion of 8.85%) and, hence, were below 22.62% and above 6.38%. The test item is considered positive and predicted to be a sensitiser (low reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 June 2019 - 02 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
June 25, 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: EC method B.60: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, Commission Regulation (EU) No 2017/735 adopted February 14, 2017, published in the Official Journal of the European Union L 112/1
Version / remarks:
April 28, 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Details on study design:
The test described in the OECD Guideline 442D is proposed to address the second key event in the Adverse Outcome Pathway (AOP) underlying skin sensitisation. Skin sensitisers are reported to induce genes that are regulated by the antioxidant response element (ARE)

Preparation of the keratinocyte cultures:
A transgenic cell line derived from HaCaT human keratinocytes having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (KeratinoSens™ cell line ). The 6th and 8th passage were used. For testing, cells were 80-90% confluent, and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.

Preparation of the test and control items:
The test item was completely dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mM. Fresh preparations of the test and control items were used for the treatment. The final concentration of the vehicle in the culture system did not affect cell viability or growth rate.Based on the stock solution of the test item, serial dilutions were made using solvent to obtain 12 master concentrations to be tested (from 0.098 to 200 mM). The master concentrations were then further diluted in treatment culture medium containing 1% serum , so that the final concentrations of the test item range from 0.98 to 2000 µM. The test item was completely dissolved in treatment culture medium to a concentration of 125 µM. Test item precipitation was noted starting at a concentration of 250 µM. The solvent DMSO was used as the negative control. Six wells per plate were prepared. It was diluted following the same dilution scheme as described for the master concentrations, so that the final negative control concentration is 1%, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.
Cinnamic aldehyde was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 µM.

Application of the test and control items:
For each test chemical and positive control item, one experiment is needed to derive a prediction (positive or negative), consisting of at least two independent repetitions each containing three replicates (i.e. n=6). Each independent repetition was performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may come from the same passage however. After seeding, cells were grown for 24 hours in the 96-well microtiter plates. The medium was then removed and replaced with fresh culture medium (150 µL culture medium containing 1% serum but without Geneticin5) to which 50 µL of the diluted test and control items were added. Three wells per plate were carried out containing no cells to assess background values. The treated plates were then incubated for about 48 hours at 37 ± 1°C in the presence of 5% CO2. Evaporation of volatile test chemicals and cross-contamination between wells by test items were avoided by covering the plates with a foil prior to the incubation with the test items.

Luciferase activity measurements:
After the 48 hour exposure time with the test and control items, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System) for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate were then placed in the luminometer (Tecan Infinite 200Pro) for reading.

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, the medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide) and cells incubated for 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed by adding 10% aqueous SDS solution to each well overnight or for up to 3 days at 37 °C. After shaking, the absorption was measured at i.e. 620 nm with a photometer.

Data evaluation:
The following parameters were calculated:
-the maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
-the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
-the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Fold luciferase activity induction was calculated, and the overall maximal fold induction (Imax) was calculated as the average of the individual repetitions.

Acceptance criteria:
The following acceptance criteria should be met.
1)the luciferase activity induction obtained with the positive control (cinnamic aldehyde), should be statistically significant above the threshold of 1.5 compared to the negative (solvent) control (e.g. using a t-test) in at least one of the tested concentrations (from 4 to 64 µM).
2)the EC1.5 value should be within two standard deviations of the historical mean. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of the positive control (cinnamic aldehyde) should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
3)the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results are discarded.

Interpretation of results and prediction model:
A prediction is considered positive if the following 4 conditions are all met in 2 of 2 or, if necessary in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1)the Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);
2)the cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration);
3)the EC1.5 value is less than (<) 1000 µM;
4)there is an apparent overall dose-response for luciferase induction (Spearman's rank correlation coefficient of p<0.01).

If in a given repetition, all of the first three conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM should also be considered as inconclusive.
In rare cases, test items which induce the luciferase activity very close to the cytotoxic levels can be positive in some repetitions at non-cytotoxic levels (i.e. EC1.5 determining concentration below (<) the IC30), and in other repetitions only at cytotoxic levels (i.e. EC1.5 determining concentration above (>) the IC30). Such test items would have been retested with more narrow dose-response analysis using a lower dilution factor (e.g. 1.33 or √2 (=1.41) fold dilution between wells), to determine if induction has occurred at cytotoxic levels or not.
Positive control results:
see table 2
Key result
Parameter:
other: fold induction of luciferase activity
Run / experiment:
1st experiment
Value:
8.72
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold induction of luciferase activity
Run / experiment:
2nd experiment
Value:
6.69
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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

Table 1: Results for the test item

 

 

Luciferase determinations

Cytotoxicity determinations

Parameter

Imax

EC1.5[µM]

IC50[µM]

IC30[µM]

Test item

Repetition 1

8.74*

12.65

335.52

166.09

Repetition 2

6.69*

17.26

233.32

144.69

Average

7.72

14.77

279.79

155.02

*= statistically significantly different as compared to the negative control

Table 2: Results for the positive control

Positive control: Induction values Reference

Criteria#

cinnamic aldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Induction 64 µM

EC1.5

Repetition 1

1.17

1.31

1.47

2.01*

2.96*

16.95

TRUE

TRUE

Repetition 2

1.12

1.19

1.37

1.88*

3.10*

20.16

TRUE

TRUE

Average

1.14

1.25

1.42

1.94

3.03

18.49

TRUE

TRUE

*= statistically significantly different compared to the negative control

# =the induction in the two replicates at 64 µM should be between 2 and 8, the EC1.5value should be between 7 µM and 30 µM.

Table 3: Historical data of negative and positive controls


(Most recent background data from GLP studies of the years 2017 and 2018)

Negative control (n = 23)

 

Luciferaseinduction

Viability [%]

 

Average induction#

CV (%)

Average viability#

Mean± SD

1.00 ± 0.08

11.10 ± 3.90

100.00 ± 5.56

Range

0.82 - 1.31

6.30 - 19.26

81.80 - 122.57

#mean value of three technical replicates (one repetition)

Positive control cinnamic aldehyde (n = 9)

Luciferase induction#

64 µM

EC1.5[µM]

2*SD

Imax

2.98 ± 1.07

20.37 ± 7.96

15.92

2.98 ± 1.07

# the average induction at 64 µM in the two repetitions should be between 2 and 8, the EC1.5value should be ideally between 7 µM and 30 µM or within 2*SD of the historical mean.

Interpretation of results:
other: activation of keratinocytes positive
Conclusions:
In this study under the given conditions the test item did 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 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.
Executive summary:

The test item was examined for sensitising properties in the ARE-Nrf2 luciferase test method (OECD TG 442D) addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes by means of quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cytotoxicity was determined with the MTT assay.

The test item was tested at 12 concentrations in the range from 0.98 to 2000 µM. The test item was completely dissolved in treatment culture medium to a concentration of 250 µM. Test item precipitation was noted macroscopically starting at a concentration of 500 µM. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.

The maximal average fold induction of the luciferase activity (Imax) values were 8.74 or 6.69 and from the dose dependent increase, EC1.5 values of 12.65 or 17.26 µM have been calculated for the first or second repetition, respectively.

The corresponding cell viability was > 70% (97.96% and 104.07%) leading to IC50 values of 335.52 or 233.32 µM in repetitions 1 and 2, respectively.

The solvent control and the positive control cinnamic aldehyde were run in all repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

A dose response for luciferase activity induction was observed for each individual repetition as well as for an overall luciferase activity induction.

The test item revealed sensitising properties in the ARE-Nrf2 Luciferase test method.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. 

DPRA test

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.85% for cysteine and 56.79% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide.

Test item-treated samples revealed a cysteine peptide depletion of 6.55% and a lysine peptide depletion of 11.14% (mean peptide depletion of 8.85%) and, hence, were below 22.62% and above 6.38%. The test item is considered positive and predicted to be a sensitiser (low reactivity) in the Direct Peptide Reactivity Assay (DPRA).

ARE-Nrf2 luciferase test method

The test item was examined for sensitising properties in the ARE-Nrf2 luciferase test method (OECD TG 442D) addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes by means of quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cytotoxicity was determined with the MTT assay.

The test item was tested at 12 concentrations in the range from 0.98 to 2000 µM. The test item was completely dissolved in treatment culture medium to a concentration of 250 µM. Test item precipitation was noted macroscopically starting at a concentration of 500 µM. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.

The maximal average fold induction of the luciferase activity (Imax) values were 8.74 or 6.69 and from the dose dependent increase, EC1.5 values of 12.65 or 17.26 µM have been calculated for the first or second repetition, respectively.

The corresponding cell viability was > 70% (97.96% and 104.07%) leading to IC50 values of 335.52 or 233.32 µM in repetitions 1 and 2, respectively.

The solvent control and the positive control cinnamic aldehyde were run in all repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

A dose response for luciferase activity induction was observed for each individual repetition as well as for an overall luciferase activity induction.

The test item revealed sensitising properties in the ARE-Nrf2 Luciferase test method.

Conclusion:

Based on the results from the in chemico and in vitro studies and considering the Adverse Outcome Pathway (AOP) the test item in predicted as 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 the test item is classified as skin sensitising Cat.1 (H317) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.