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Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2018 to 02 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The objective of this study was to determine the reactivity of the teat material towards model synthetic peptides containing either cysteine or lysine, and to categorise the test material in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers.
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 °C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are 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 non-sensitisers.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-isobutylbutyl acetate
EC Number:
233-588-4
EC Name:
3-methyl-1-isobutylbutyl acetate
Cas Number:
10250-45-0
Molecular formula:
C11H22O2
IUPAC Name:
3-methyl-1-isobutylbutyl acetate
Test material form:
liquid
Details on test material:
- Physical Description: Clear colourless liquid
- Storage Conditions: At room temperature

In vitro test system

Details on the study design:
DOSE FORMULATION AND ANALYSIS
- Preparation of Test Material: 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. An appropriate solvent dissolved the test material completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: Acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol (EtOH) and methanol (MeOH).
- Test material stock solutions were prepared freshly for each reactivity assay.
- For both the cysteine and lysine reactivity assay 34.49 mg of test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1851 µL ACN 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. Any residual volumes were discarded.
 
TEST SYSTEM
- 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.  
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Storage: The peptides were stored in the freezer (≤ 15 °C) for a maximum of 6 months.
 
EXPERIMENTAL DESIGN
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 10.4 mg of SPCC in 20.76 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- 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.
- SPCC Calibration Curve: A SPCC calibration curve was prepared as follows:
STDcys1: 0.534 mM (1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN)
STDcys2: 0.267 mM (1 mL STDcys1 + 1 mL STDcys7)
STDcys3: 0.133 mM (1 mL STDcys2 + 1 mL STDcys7)
STDcys4: 0.067 mM (1 mL STDcys3 + 1 mL STDcys7)
STDcys5: 0.033 mM (1 mL STDcys4 + 1 mL STDcys7)
STDcys6: 0.017 mM (1 mL STDcys5 + 1 mL STDcys7)
STDcys7: 0 mM (8 mL phosphate buffer (pH 7.5) + 2 mL ACN)
- 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) (n=1): CCcys-209427/A (750 µL Phosphate buffer pH 7.5, 200 µL ACN and  50 µL 209427/A test solution (100 mM)).
Cinnamic aldehyde (PC) (n=3): PCcys-1 to PCcys-3 (750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN and 50 µL Cinnamic aldehyde solution (100 mM in ACN)).
Test material 209427/A (n=3): 209427/A-cys-1 to 209427/A-cys-3 (750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN and 50 µL 209427/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 11.3 mg of SPCL in 21.81 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.
- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as follows:
STDlys1: 0.534 mM (1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN)
STDlys2: 0.267 mM (1 mL STDlys1 + 1 mL STDlys7)
STDlys3: 0.133 mM (1 mL STDlys2 + 1 mL STDlys7)
STDlys4: 0.067 mM (1 mL STDlys3 + 1 mL STDlys7)
STDlys5: 0.033 mM (1 mL STDlys4 + 1 mL STDlys7)
STDlys6: 0.017 mM (1 mL STDlys5 + 1 mL STDlys7)
STDlys7: 0 mM (8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN)
- 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) (n=1): CClys-209427/A (750 µL Ammonium acetate buffer pH 10.2 and 250 µL 209427/A test solution (100 mM)).
Cinnamic aldehyde (PC) (n=3): PClys-1 to PClys-3 (750 µL Stock solution of 0.667 mM SPCL and 250 µL Cinnamic aldehyde solution (100 mM in ACN)).
Test material 209427/A (n=3): 209427/A-lys-1 to 209427/A-lys-3 (750 µL Stock solution of 0.667 mM SPCL and 250 µL 209427/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 25.5 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. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and transferred to a new vial.
 
HPLC-PDA Analysis
- SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following systems:
- System 1 (used for Cysteine Reactivity Assay):
Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
LC Column oven 300 (Thermo Scientific)
Surveyor PDA detector (Thermo Scientific)

- System 2 (used for Lysine Reactivity Assay):
Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
Column Oven #151006 (Grace, Worms, Germany)
Surveyor PDA detector (Thermo Scientific)
 
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:
- The standard calibration curve had to have an r^2 > 0.99.
- 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.
- 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.
- The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
- 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:
- 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.
- 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 %  
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:
0 % ≤ Mean % depletion ≤ 6.38 %: No or minimal reactivity = NEGATIVE
6.38 % < Mean % depletion ≤ 22.62 %: Low reactivity = POSITIVE
22.62 % < Mean % depletion ≤ 42.47 %: Moderate reactivity = POSITIVE
42.47 % < Mean % depletion ≤ 100 %: High reactivity = POSITIVE

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
mean
Parameter:
other: SPCC depletion (%)
Value:
2.1
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: ± SD 3.7 %
Key result
Run / experiment:
mean
Parameter:
other: SPCL depletion (%)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: ± SD 0.0 %
Key result
Run / experiment:
mean
Parameter:
other: Mean of SPCC and SPCL depletion (%)
Value:
1.1
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Other effects / acceptance of results:
SOLUBILITY OF THE TEST MATERIAL
- At a concentration of 100 mM, the test material was not soluble in MQ and ACN:MQ (1:1, v/v), but was soluble in ACN, IPA, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), EtOH and MeOH. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test material in this 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 25.5 hours of incubation at 25 ± 2.5 °C.
- Acceptability of the Cysteine Reactivity Assay: The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.998. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.513 ±0.004 mM while the mean peptide concentration of Reference Controls C was 0.504 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test material 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 1.5 %. 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 17.26. The mean A220/A258 ratio ± 10 % range was 15.54-18.99. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.0 % ± 0.2 %. This was within the acceptance range of 60.8 % to 100 % with a SD that was below the maximum (SD <14.9 %).
- Results Cysteine Reactivity Assay for the Test Material: Preparation of a 100 mM Alicate stock solution in ACN 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. Upon preparation and after incubation a precipitate was observed in the co-elution control (CC) and test material samples. In this case one cannot be sure how much test material remained in the solution to react with the peptide.
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 209427/A-cys samples, the mean SPCC A220/A258 area ratio was 17.29. Since this was within the 15.54-18.99 range, this again indicated that there was no co elution of the test material with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test material was 2.1 % ± 3.7 %.

LYSINE REQCTIVITY ASSAY
- The reactivity of Alicate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 25.5 hours of incubation at 2 5± 2.5 °C.
- Acceptability of the Lysine Reactivity Assay: The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.482 ± 0.022 mM while the mean peptide concentration of Reference Controls C was 0.457 ± 0.034 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) 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 5.2 %. 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 16.55. The mean A220/A258 ratio ± 10 % range was 14.89-18.20. 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 48.3 % ± 3.1 %. This was within the acceptance range of 40.2 % to 69.0 % with a SD that was below the maximum (SD <11.6 %).
- Results Lysine Reactivity Assay for the Test Material: Preparation of a 100 mM test material stock solution in ACN 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. Upon preparation and after incubation a precipitate was observed in the CC and test material samples. In this case one cannot be sure how much test material remained in the solution to react with the peptide.
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 209427/A-lys samples, the mean SPCL A220/A258 area ratio was 16.33. Since this was within the 14.89-18.20 range, this again indicated that there was no co-elution of the test material with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Material was 0.0 % ± 0.0 %.

DPRA PREDICTION AND REACTIVITY CLASSIFICATION
- Upon preparation and after incubation of the SPCC and SPCL test material samples, a precipitate was observed.
- In the cysteine reactivity assay the test material showed 2.1 % SPCC depletion while in the lysine reactivity assay the test material showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1 % and as a result the test material was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test material remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Any other information on results incl. tables

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Material

 

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

Test material

2.1 %

± 3.7 %

0.0 %

± 0.0 %

1.1 %

Negative: No or minimal reactivity

SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:
other: Negative in the DPRA Assay
Conclusions:
Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with 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 non-sensitisers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

Upon preparation and after incubation of the SPCC and SPCL test material samples, a precipitate was observed.

In the cysteine reactivity assay the test material showed 2.1 % SPCC depletion while in the lysine reactivity assay the test material showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1 % and as a result the test material was 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.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test material was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test material remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).