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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-24 to 2018-11-13
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))
Version / remarks:
4 February 2015
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

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
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.

Number of replicates: co-elution controls, reference controls and samples were measured in triplicates.
SPCC 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 ACN. In addition, a RCcysCDMSO:ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCDMSO:ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL DMSO:ACN (1:9, v/v).

Co-elution control (CC): 750 µL Phosphate buffer pH 7.5, 200 µL ACN, 50 µL CCys 209631/A solution (100 mM).

Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL Cinnamic aldehyde solution (100 mM in ACN).

Test item 209629/A: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL 209631/A test solution (100 mM).
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 RClysCDMSO:ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCDMSO:ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL DMSO:ACN (1:9, v/v).

Co-elution control (CC): 750 µL Ammonium acetate buffer pH 10.2, 250 µL CClys 209631/A test solution (100 mM)

Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCL, 250 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item 209629/A: 750 µL Stock solution of 0.667 mM SPCL, 250 µL 209631/A test solution (100 mM)

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 23.5 h and 25 h, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 h.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation.
At a concentration of 100 mM, TA-3 was not soluble in ACN, MQ, ACN:MQ (1:1, v/v), isopropanol and acetone:ACN (1:1, v/v), but was soluble in DMSO:ACN (1:9, v/v). Therefore this solvent was used to dissolve the test item in this DPRA study.
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
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

Results and discussion

Positive control results:
% depletion cystein peptide: mean 68.9%; SD 0.3%
% depletion lysine peptide: mean 60.5%; SD 2.1%

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean percent cysteine and lysine depletion
Value:
15.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reactivity
Key result
Parameter:
other: mean percent depletion
Run / experiment:
cysteine peptide
Value:
1.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: mean percent depletion
Run / experiment:
lysine peptide
Value:
30.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reactivity
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

Acceptability of the Cysteine Reactivity Assay
The correlation coefficient (r²) of the SPCC standard calibration curve was 0.998. Since the r² was >0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.518 ± 0.005 mM, the mean peptide concentration of Reference Controls C was 0.518 ± 0.006 mM and the mean peptide concentration of Reference Controls CDMSO:ACN was 0.474 ± 0.012 mM. The means of Reference Control samples A, C and CDMSO:ACN were all within the acceptance criteria of 0.50 ± 0.05 mM. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.2%. 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 18.00. The mean A220/A258 ratio ± 10% range was 16.20-19.80. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. 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 68.9% ± 0.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay
The correlation coefficient (r²) of the SPCL standard calibration curve was 0.998. Since the r² was >0.99, the SPCL standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.511 ± 0.018 mM, the mean peptide concentration of Reference Controls C was 0.532 ± 0.013 mM and the mean peptide concentration of Reference Controls CDMSO:ACN was 0.511 ± 0.018 mM. The means of Reference Control samples A, C and CDMSO:ACN were all within the acceptance criteria of 0.50 ± 0.05 mM. The CV of the peptide areas for the nine Reference Controls B and C was 2.2%. 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 16.85. The mean A220/A258 ratio ± 10% range was 15.17-18.54. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.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 60.5% ± 2.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). 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 60.5% ± 2.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Any other information on results incl. tables

Upon preparation of the SPCC test item samples a precipitate was observed while upon preparation of the SPCL test item samples no precipitate or phase separation was observed. After incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 1.2% SPCC depletion while in the lysine reactivity assay the test item showed 30.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 15.8% and as a result the test item was positive in the DPRA and was classified in the “lowreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. In conclusion, this DPRA test is valid. TA-3 was positive in the DPRA and was classified in the “low reactivity class” when using theCysteine 1:10 / Lysine 1:50 prediction model.

Applicant's summary and conclusion

Interpretation of results:
other: positive
Conclusions:
In conclusion, the test item was tested for skin sensitisation in the Direct Peptide Reactivity Assay. This DPRA test is considered valid, thus, the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.