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:
15 January 2018 - 18 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

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
Justification for non-LLNA method:
A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Storage condition of test material: At room temperature

In chemico test system

Details on study design:
TEST ITEM PREPARATION
Solubility of the test substance in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test substance 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, acetone/ACN (1:1, v/v), dimethylsulfoxide (DMSO)/ACN (1:9, v/v) and methanol.
Test substance stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 56.95 mg of test substance was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1622 µL ACN/MQ (1:1, v/v) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test substance was dissolved. The test substance, 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. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test substance 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.

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

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))]x100

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 substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 1), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Mean SPCC depletion(%)
Run / experiment:
Cysteine Reactivity Assay
Value:
1.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 1.6%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 78.7% ±1.1%
Remarks on result:
other: SD: 1.0%
Parameter:
other: Mean SPCL depletion (%)
Run / experiment:
Lysine Reactivity Assay
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 3.7%
Positive controls validity:
valid
Remarks:
Mean percentage SPCL: 59 ± 1.3%
Remarks on result:
other: SD: 0.0%
Other effects / acceptance of results:
Upon preparation of the SPCC test item samples, a precipitate was observed while no precipitate was observed in the SPCC test item samples after incubation.
Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

See Table 2 & 3 "any other information on results incl. tables" for acceptibility criteria.

Any other information on results incl. tables

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.998

>0.99

0.997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.501 ± 0.003

0.50 ± 0.05

0.509 ± 0.010

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.496 ± 0.006

0.50 ± 0.05

0.518 ± 0.004

Mean peptide concentration RC-CACN/MQsamples (mM)

0.50 ± 0.05

0.495 ± 0.001

0.50 ± 0.05

0.497 ± 0.002

CV (%) for RC samples

B and C

<15.0

1.6

<15.0

3.7

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

78.7

40.2-69.0

59.1

SD of peptide depletion cinnamic aldehyde (%)

<14.9

1.1

<11.6

1.3

SD of peptide depletion for the test substance (%)

<14.9

1.0

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

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 substance

1.8%

±1.0%

100.0%

±0.0%

50.9%

Positive: High reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:
other: Study cannot be used for classification independetly, but in a WoE for the end point Skin Sensitisation
Conclusions:
The test substance was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers.

Following incubation of the test substance 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 the prediction model.

 ACN/MQ (1:1, v/v) was found to be an appropriate solvent to dissolve the test substance, and was therefore used in this DPRA study. Cinnamic aldehyde was used as a positive control. 

Upon preparation of the SPCC test item samples, a precipitate was observed while no precipitate was observed in the SPCC test item samples after incubation. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

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 substance, were within the acceptability criteria for the DPRA assay. Therefore, the study was considered to be valid. No co-elution of the test item with SPPC or SPCL was observed.

 

In the cysteine reactivity assay the test substance showed 1.8% SPCC depletion while in the lysine reactivity assay the test substance showed 100.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.9% and as a result the test substance 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 substance was considered to be positive in the DPRA.