Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2018 to 08 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: white powder
- Storage conditions: at room temperature
Specific details on test material used for the study:
No correction for the purity/composition of the test material was performed.
Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. The following solvents were evaluated: acetonitrile (ACN) and Milli-Q water (MQ).

In chemico test system

Details on study design:
- Dose formulation
Test material stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 34.87 mg of test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1762 μL MQ after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test material was dissolved. The test material, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

- Test system
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL (JPT Peptide Technologies GmbH, Berlin, Germany)

Preparation of Solutions for Cysteine Reactivity Assay
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL acetonitrile (ACN). In addition, a RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL cosolvent.
- A SPCC calibration curve was prepared
- Co-elution Control, Test Material and Positive Control Samples
The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared, as follows:
* Co-elution control (CC) - 1 replicate - (CCcys-209202/A): 750 μL Phosphate buffer pH 7.5, 200 μL ACN, 50 μL 209202/A test solution (100 mM)
* Cinnamic aldehyde (PC) - 3 replicates - (PCcys-1 to PCcys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL Cinnamic aldehyde solution (100 mM in ACN)
* Test mateiral 209202/A - 3 replicates - (209202/A-cys-1 to 209202/A-cys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL 209202/A test solution (100 mM)

Preparation of Solutions for Lysine Reactivity Assay
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL
cosolvent.
- SPCL Calibration Curve
A SPCL peptide calibration curve was prepared
- Co-elution Control, Test Material and Positive Control Samples
The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared, as follows:
* Co-elution control (CC) - 1 replicate - (CClys-209202/A): 750 μL Ammonium acetate buffer pH 10.2, 250 μL 209202/A test solution (100 mM)
* Cinnamic aldehyde (PC) - 3 replicates - (PClys-1 to PClys-3): 750 μL Stock solution of 0.667 mM SPCL, 250 μL Cinnamic aldehyde solution (100 mM in ACN)
* Test mateiral 209202/A - 3 replicates - (209202/A-lys-1 to 209202/A-lys-3): 750 μL Stock solution of 0.667 mM SPCL, 250 μL 209202/A test solution (100 mM)

> Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test material samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 26 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

> HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA:
Mobile phase A: 0.1 % (v/v) TFA in Milli-Q water B: 0.085 % (v/v) TFA in ACN
Gradient
Cysteine :
0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5 min 10 % B
20 min 10 % B

Lysine:
0 min 10% B
10 min 20% B
11 min 90% B
13 min 90% B
13.5 min 10% B
20 min 10% B

Flow: 0.35 mL/min
Injection volume: 3 μL
Sample tray temperature: Set at 25 °C
Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
Column temperature: Set at 30 °C
Detection: Photodiode array detection, monitoring at 220 and 258 nm

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r² > 0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8 and 100 % for SPCC and between 40.2 and 69.0 % for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be < 14.9 % for the Percent Cysteine Peptide Depletion and < 11.6 % for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be < 15.0 %.
The following criteria had to be met for a test material’s results to be considered valid:
a) The maximum SD for the test material replicates had to 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 had to be 0.50 ± 0.05 mM.

ANALYSIS
- Data Evaluation
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1 − (Peptide Peak Area in Replicate Injection (at 220 nm) / Mean Peptide Peak Area in Reference Controls (at 220 nm))] × 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90 % < mean area ratio of control samples < 110 % gives a good indication that co-elution has not occurred. However, calculation of peak purity (area ratio 220 nm/258 nm) might not always be possible, particularly if the test material is highly reactive with the peptide.

- Data Interpretation
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test material. 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 a skin sensitiser and a non-sensitiser:
Negative: 0 % ≤ Mean % depletion ≤ 6.38 % (No or minimal reactivity)
Positive: 6.38 % < Mean % depletion ≤ 22.6 2% (Low reactivity), 22.62 % < Mean % depletion ≤ 42.47 % (Moderate reactivity) and 42.47 % < Mean % depletion ≤ 100 % (High reactivity)

Results and discussion

Positive control results:
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 75.3 ± 2.1 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD < 14.9 %).
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 55.5 ± 2.4 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD <11.6 %).

In vitro / in chemico

Results
Key result
Parameter:
other: Mean of SPCC and SPCL depletion
Run / experiment:
DPRA Prediction
Value:
50.1
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- Solubility Assessment of the Test Material
At a concentration of 100 mM, the test material was not soluble in acetontrile (ACN), but was soluble in Milli-Q water (MQ). Therefore this solvent was used to dissolve the test material in this DPRA study.

- Cysteine Reactivity Assay
The reactivity of the test material towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 26 hours of incubation at 25 ± 2.5 °C.

The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCC Depletion. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 5.8 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 19.28. The mean A220/A258 ratio ± 10% range was 17.35-21.20. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Preparation of a 100 mM test material stock solution in MQ showed that the test material was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test material samples were visually inspected. No precipitate was observed in any of the samples after preparation, however, after incubation a precipitate (a gelatinous layer on the bottom of the vial) was observed in the co-elution control (CC) and test material samples. In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test material with SPCC. For the 209202/A-cys samples, the mean SPCC A220/A258 area ratio could not be determined. Since the test material displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm.
Overall, it can be concluded that the test material did not co-elute with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls CMQ. The mean Percent SPCC Depletion for the test material was 100 ± 0.0 %.

- Lysine Reactivity Assay
The reactivity of the test material towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 26 hours of incubation at 25 ± 2.5 °C.

The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.994. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent
SPCL Depletion. The CV of the peptide areas for the nine Reference Controls B and C was 2.5 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 15.65. The mean A220/A258 ratio ± 10 % range was 14.08-17.21. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Preparation of a 100 mM test material stock solution in MQ showed that the test material was dissolved completely. Upon preparation and after incubation, both the CC as well as the test material samples were visually inspected. No precipitate was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test material with SPCL. For the 209202/A-lys samples, the mean SPCL A220/A258 area ratio was 15.32. Since this was within the 14.08 - 17.21 range, this again indicated that there was no coelution of the test material with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls CMQ. The mean Percent SPCL Depletion for the test material was 0.2 ± 0.3 %.

> DPRA Prediction and Reactivity Classification
Upon preparation of the SPCC and SPCL test material samples, no precipitate was observed in any of the samples, however, after incubation, a precipitate was observed for the SPCC test material samples.
In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Therefore, the test material was considered to be positive in the DPRA.

Applicant's summary and conclusion

Interpretation of results:
other: Positive DPRA prediction
Conclusions:
In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Therefore, the test material was considered to be positive in the DPRA.
Executive summary:

The reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

During the study, after incubation of the test materialwith either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and nonsensitisers.

In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Therefore, the test material was considered to be positive in the DPRA.