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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 31, 2017 to February 05, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Protocol No. 154 (Direct Peptide Reactivity Assay for Skin Sensitisation Testing)
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In chemico test system

Details on study design:
See below 'Any other information for materials and methods incl. tables' for details on study design.

Test and reference substances

Test substance
TPhysical State: Light Amber liquid
Storage Condition: Room Temp.
Solubility: Water, Ethanol.
Solvent used: Isopropanol
Concentration tested: 100mM


Reference Substance
Supplier: Sigma-Aldrich
Reference Substance Name: Cinnamic Aldehyde
Lot Number: MKBV4784V
CAS Number: 104-55-2
Purity: >95%
Concentration Tested: 100mM
Solvent: HPLC Grade Acetonitrile (CAS No. 75-05-8)
Expiry Date: Apr 2020
Storage Conditions: Positive control is prepared fresh on Day 1 of main test from stock chemical stored at room temperature

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: % Cysteine Peptide Depletion
Run / experiment:
1
Value:
ca. 0.892
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: % Lysine Peptide Depletion
Run / experiment:
2
Value:
ca. 2.306
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: Mean % Peptide Depletion (Cys + Lys)
Run / experiment:
3
Value:
ca. 1.599
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No or minimal reactivity
Other effects / acceptance of results:
Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

Any other information on results incl. tables

Results

 

Solvent Selection

Prior to the main test, the test substance was assessed for solubility and was found to be soluble in Isopropanol at 100mM.

 

Acceptance criteria

Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

 

Criterion

Run 1 (Cysteine)

Run 2 (Lysine)

Outcome

Std Curve r2 >0.99

0.997

0.991

PASS

PC 60.8% to 100% depletion Cys

64.698

N/A

PASS

PC 40.2% to 69.0% depletion Lys

N/A

52.256

PASS

SDCys Depletion PC<14.9%

10.909

N/A

PASS

SDLys Depletion PC<11.6%

N/A

0.991

PASS

RefA Mean Conc 0.50 ± 0.05mM

0.556*

0.533

PASS*/PASS

Peak Area CV RefB <15.0%

8.970

0.377

PASS

Peak Area CV RefC <15.0%

5.137

0.844

PASS

SDCys Depletion Test Substance <14.9%

1.546

N/A

PASS

SDLys Depletion Test Substance <11.6%

N/A

0.332

PASS

RefC Mean Conc 0.50 ± 0.05mM

0.547

0.528

PASS

Cys = Cysteine, Lys = Lysine, SD = Standard Deviation, CV = Coefficient of Variation, PC = Positive Control. *Passed as acceptable by Study Director.

 

Results Summary

The test substance produced 1.599% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. A single HPLC analysis for both the Cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. No co-elution (peak areas produced at 220nm with the same retention time as the peptide) was observed for this test substance.

 

Name

Test Substance ID

% Cysteine Peptide Depletion

% Lysine Peptide Depletion

Mean % Peptide Depletion (Cys + Lys)

DPRA Prediction

 

DPRA

Reactivity Class

Acetamide MEA

TA4

0.892

2.306

1.599

Non-Sensitiser

No or Minimal Reactivity

 

Deviations

During the initial runs there was an HPLC system failure and therefore the study was halted (data was retained) and the study was repeated once a new system had been installed and validated.

Conclusion

The final mean % peptide depletion observed in the DPRA was 1.599%. Therefore, Acetamide MEA was classified as a non-sensitiser with no or minimal reactivity as per the Cysteine 1:10 / Lysine 1:50 prediction model.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified based on EU CLP Criteria
Conclusions:
Under study conditions, the test substance was determined to be non-sensitiser to the skin.
Executive summary:

A study was conducted to determine the skin sensitisating potential of the test substance using Direct Peptide Reactivity Assay (DPRA), according to OECD Guideline 442C, in compliance with GLP. Test substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an high performance liquid chromatography (HPLC) system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. Test substance was compared to vehicle controls containing the test substance solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes. The test substance produced 1.599% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. A single HPLC analysis for both the cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance was concluded to be non-sensitiser to the skin (XCellR8, 2018).