N-2-hydroxyethylacetamide

Skin sensitisation

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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 reactivity assay (DPRA)

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

Test material information

1

Reference substance name:

Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

Molecular formula:

C4H9NO2 (amide) & C6H11NO3 (amido ester)

IUPAC Name:

Reaction mass of N-2-hydroxyethylacetamide and N,O-diacetyl-2-aminoethanol

Test material form:

liquid

In chemico test system

Details on the 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

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: % Cysteine Peptide Depletion

Value:

0.892

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: % Lysine Peptide Depletion

Value:

2.306

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 3

Parameter:

other: Mean % Peptide Depletion (Cys + Lys)

Value:

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 r^2>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).