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Description of key information

Based on a weight of evidence evaluation of the available data from three in vitro and in chemico assays covering two of three key events, the test item is considered to not be a skin sensitiser:

- DPRA: negative
- Keratinosens: 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:
2020-01-28
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))
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Skin sensitisation (In chemico test system)

Formulation of the Test Item
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.1479 g ± 10 %) based on the molecular weight and purity of the substance with the equation below. 0.1479 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.1478 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the runs.

HPLC conditions:
HPLC: Shimadzu LC-2030i Prominence
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7μL
System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.100 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Positive control: CINNAMALDEHYDE

Key result
Run / experiment:
other: mean out of three replicates
Parameter:
other: mean peptide depletion cysteine
Value:
9.68
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: mean out of three replicates
Parameter:
other: mean peptide depletion lysine
Value:
0.02
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

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 peptide depletion values for the positive control and the test item

Name, replicate number

Obtained mean

% cysteine
peptide depletion

Obtained mean
% Lvsine peptide
depletion

Mean %
obtained peptide
depletion

test item

9.68

0.02

4.85

CINNAMALDEHYDE

73.67

62.24

67.96

The average percent peptide depletion was calculated for the test item. Since no co-elution was observed, applying the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. The mean percent peptide depletion value was 4.85 %. Thus, the test item is considered to be negative in the DPRA and classified in the no or minimal reactivity class when using the cysteine 1:10 / lysine 1:50 prediction model.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these results a direct peptide reactivity assay (DPRA) according to OECD 442C the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the study.
Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion resulted from the positive control cinnamaldehyde was 73.67 % ± 0.94 % with cysteine peptide and 62.24 % ± 3.78 % with the lysine peptide.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.0 % and 0.2 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.68 % ± 2.26 % while the percent lysine peptide depletion was 0.02 % ± 0.44 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 4.85 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. It is thus not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019-12-04
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)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
Skin sensitisation (in vitro test system)

Procedure of the KeratinoSens™ method
0. Preincubation of cells
1. Seeding of cells for testing - 24 h incubation
2. Solubility testing Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment

Formulation of the Test Item
Based on the test item stock solutions made of sterile ultrapure water, that was sterilized by filtering via 0.22 μm membrane filter, 2-fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25-fold into exposure medium to obtain the 4 × master plate, by adding 10 μL of the 100 × master concentrations to 230 μL exposure medium and correcting DMSO concentration by adding 10 μL DMSO to all twelve 4 × master solutions.

Preparation of cells
Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.

Exposure
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4× master solution to 150 μL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.

Luciferase activity measurements
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 μL), and 1× lysis buffer (20 μL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.
Key result
Run / experiment:
other: based on two runs
Parameter:
other: Statistically significant induction over 1.5-fold
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: based on two runs
Parameter:
other: Viability ≥ 70 % at lowest concentration with ≥ 1.5-fold
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: based on two runs
Parameter:
other: EC1.5 (μM)
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, our laboratory demonstrated technical proficiency (Study Number: 392-442-4012), using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory.

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
Interpretation of results:
GHS criteria not met
Conclusions:
Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.

Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold. Therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.

Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

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

There are two of three in vitro/in chemico studies available with the test item assessing its skin sensitising potential. The studies were conducted according to GLP and the respective OECD TG (OECD 442C, OECD 442D) and are both considered valid. The available data is considered suitable and reliable to allow assessment of the test items properties in regards to skin sensitisation.

OECD 442 C (DPRA)

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion resulted from the positive control cinnamaldehyde was 73.67 % ± 0.94 % with cysteine peptide and 62.24 % ± 3.78 % with the lysine peptide.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.0 % and 0.2 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.68 % ± 2.26 % while the percent lysine peptide depletion was 0.02 % ± 0.44 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 4.85 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. It is thus not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

OECD 442 D (KeratinoSense)

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.

Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold. Therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.

Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Conclusion

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

 

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

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012; Table 3). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

Table 3: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA

LuSens/

KeratinoSensTM

MUSST/

h-CLAT

Test battery evaluation

positive

positive

positive

sensitizer

positive

positive

negative

sensitizer

positive

negative

positive

sensitizer

positive

negative

negative

non-sensitizer

negative

positive

positive

sensitizer

negative

positive

negative

non-sensitizer

negative

negative

positive

non-sensitizer

negative

negative

negative

non-sensitizer

 

In accordance with the published evaluation scheme (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is considered to be a skin sensitizer based on the following results:

- DPRA: negative
- Keratinosens: negative

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitising properties, the test item does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 (CLP).