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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

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

Formulation of the Test Item
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.1479 g ± 10 %) based on the molecular weight and purity of the substance with the equation below. 0.1479 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.1478 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the runs.

HPLC conditions:
HPLC: Shimadzu LC-2030i Prominence
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7μL
System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.100 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Positive control: CINNAMALDEHYDE

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean peptide depletion cysteine
Run / experiment:
mean out of three replicates
Value:
9.68
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion lysine
Run / experiment:
mean out of three replicates
Value:
0.02
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1 Mean peptide depletion values for the positive control and the test item

Name, replicate number

Obtained mean

% cysteine
peptide depletion

Obtained mean
% Lvsine peptide
depletion

Mean %
obtained peptide
depletion

test item

9.68

0.02

4.85

CINNAMALDEHYDE

73.67

62.24

67.96

The average percent peptide depletion was calculated for the test item. Since no co-elution was observed, applying 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. The mean percent peptide depletion value was 4.85 %. Thus, the test item is 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these results a direct peptide reactivity assay (DPRA) according to OECD 442C the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the study.
Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion resulted from the positive control cinnamaldehyde was 73.67 % ± 0.94 % with cysteine peptide and 62.24 % ± 3.78 % with the lysine peptide.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.0 % and 0.2 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.68 % ± 2.26 % while the percent lysine peptide depletion was 0.02 % ± 0.44 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 4.85 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. It is thus not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.