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Skin sensitisation

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Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 September 2017 - 20 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The DPRA assay 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:
2017
Report Date:
2017

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 (incl. certificate)
Remarks:
03 November 2015
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Physical appearance: colourless to pale yellow liquid
Storage conditions: at room temperature protected from light, container flushed with nitrogen.

In chemico test system

Details on study design:
TEST ITEM PREPERATION
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item 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), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay. For the cysteine and lysine reactivity assay, respectively, 28.39 mg and 27.54 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1629 μL and 1581 μL ACN, respectively, to obtain 100 mM solutions. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, 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 of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: 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 time between placement of the samples in the autosampler and analysis of the first RCcysB- and RClysB-sample were 24.5 hours and 23 hours, 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 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%

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.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. 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 sensitizer and a non-sensitizer.

Results and discussion

Positive control results:
The positive control had a mean SPCC depletion of 75.4 ± 2.5% and a mean SPCL depletion of 49.2 ± 0.8%.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: SPCC mean depletion (%)
Run / experiment:
Cysteine Reactivity Assay
Value:
0.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Remarks:
SD: 1.6%
Key result
Parameter:
other: SPCL mean depletion (%)
Run / experiment:
Lysine Reactivity Assay
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Since precipitation was observed upon addition of the test item to the SPCL peptide solution, one cannot be sure how much test item remained in the solution to react with the SPCL peptide. The negative result is therefore uncertain and should be interpreted with due care.

In both the cysteine reactivity assay and the lysine reactivity assay, all acceptability criteria were met and the assays are considered valid (see table 4).

Any other information on results incl. tables

Table 4 Acceptability of the DPRA assay

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.994

>0.99

0.995

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.505 ± 0.007

0.50 ± 0.05

0.504 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

 0.494 ± 0.009

0.50 ± 0.05

0.494 ± 0.005

CV (%) for RC samples B and C

<15.0

2.0

<15.0

2.0

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

75.4

40.2-69.0

49.2

SD of peptide depletion cinnamic aldehyde (%)

<14.9

2.5

<11.6

0.8

SD of peptide depletion for Florantone T (%)

<14.9

1.6

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

* For calculation of the RC-C mean and the CV, the RCcysC-3 value was excluded (outlier due to injection error). As a result, no SD could be calculated for the mean of the RCcysC.

Table 5 SPCC and SPCL depletion and reactivity classification for Florantone T

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

Reactivity class

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

Florantone T

0.9%

±1.6%

0.0%

±0.0%

0.5%

Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
Florantone T was negative in a DPRA performed according to OECD guideline 442C and GLP principles. The test item was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The study cannot be used stand-alone to draw a conclusion on classification.