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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 116B2018
- Expiration date of the lot/batch:
- Purity test date: 15 November, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Material was soluble in isopropyl alcohol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was prepared in isopropyl alcohol.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel: 100 mM.

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system) - Details on study design: The objective of the study was to determine the skin sensitization potential of the test article based on the depletion of cysteine and/or lysine peptides following 24 hours of incubation at 25 C. Relative peptide concentration was measured by high performance liquid chromotagraphy (HPLC) with gradient elution and ultraviolet detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allowed the assigning of the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Results and discussion

Positive control results:
The percent cysteine depletion values for the positive control sample replicates ranged from 70.2 to 71.3%. The percent lysine depletion values for the positive control sample replicates ranged from 63.6 to 66.0%. The mean percent cysteine and lysine depletion values for the respective positive control samples were 70.7 ± 0.6% (N = 3; CV = 0.816%) and 64.5 ± 1.3% (N = 3; CV = 2.06%), respectively. The mean percent cysteine and lysine depletion values for the respective positive control samples were in the range allowed by the OECD guideline (1). Precipitate was not present in the cysteine positive control samples upon initial preparation (i.e. 0 hours) and following approximately 24 hours of incubation. Precipitate was not present in the lysine positive control samples upon initial preparation (i.e. 0 hours) but was present following approximately 24 hours of incubation.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Cysteine Depletion
Remarks:
Cysteine Depletion
Value:
ca. 1.88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Cysteine Depletion Percentage
Remarks:
Thy Cysteine Depletion value was 1.88% compared to the negative control.
Key result
Parameter:
other: Lysine Depletion
Remarks:
Lysine Depletion
Value:
ca. 0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Lysine Depletion Percentage
Remarks:
The lysine depletion percentage was 0.0% compared to controls.
Other effects / acceptance of results:
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: The HPLC/UV system suitability assay is considered to be valid if the following conditions are met: a.) the calibration curve should have an r2 >0.99; b.) the mean peptide concentration of reference controls (Set A) should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in ACN should be < 15.0%; c.) the mean percent depletion values of the three positive control replicates should be between 60.8% and 100% with a standard deviation (SD) as of < 14.9% for the cysteine peptide and between 40.2% and 69.0% with an SD of < 11.6% for the lysine peptide. The test chemical data should be considered to be valid if the following criteria are met: a.) the mean peptide concentration of the reference controls (Set C) for the appropriate solvent used should be 0.50 ± 0.05 mM; b.) the SD for the percent depletion values of the three test substance replicates should be < 14.9% for the cysteine peptide and < 11.6% for the lysine peptide. Negative depletion is considered as “0%” when calculating the mean.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study, the test article is considered to have no or minimal cysteine and lysine reactivity and a negative DPRA prediction.
Executive summary:

The skin sensitization potential of the test article was evaluated in the Direct Peptide Reactivity Assay (DPRA). The assay was conducted according to OECD guideline 442C. The study was performed in compliance with OECD GLP (1997). The test article was prepared at concentration of 100 mM in isopropyl alcohol and 50 uL of the test material solution was incubated for 24 hours at 25 degrees C with 750 uL of the stock cysteine peptide solution. Similarly, 250 uL of the test article solution was incubated for 24 hours at 25 degrees C with 750 uL of lysine peptide stock solution. Appropriate positive (100 mM cinnamic aldehyde) and negative (100 mM Ammonium Acetate Buffer) controls were run in parallel.  Following incubation, relative peptide concentrations were measured by high performance liquid chromotagraphy (HPLC) with gradient elution and ultraviolet detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated compared to negative control values and used in a prediction model which allowed the assigning of the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. The mean percent cysteine depletion for the test substance assay samples was 1.88 ± 0.57% (N = 3; CV = 30.3%).  The mean percent lysine depletion for the test substance assay samples was 0.00 ± 0.00% (N = 3).  Precipitate was not present in the cysteine test substance assay samples upon initial preparation of both test substance solutions (i.e. 0 hours) and following approximately 24 hours of incubation. Precipitate was present in the lysine test substance assay samples upon initial preparation of both test substance solutions (i.e. 0 hours) and following approximately 24 hours of incubation. Based on the results of the study, the test article is considered to have no or minimal cysteine and lysine reactivity and a negative DPRA prediction.