N-acetyl-β-D-glucosamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th July 2019 - 14th October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay

Justification for non-LLNA method:

This assay measures the ability of the test item to cause skin sensitisation. DPRA assay uses synthetic heptapeptides, i.e., Cysteine and Lysine, which are known for their reliability and reproducibility in a direct peptide reactivity assay and are also recommended by the OECD and other regulatory authorities.

Test material

Test material information

Constituent 1

Reference substance name:

N-acetyl-β-D-glucosamine

EC Number:

231-368-2

EC Name:

N-acetyl-β-D-glucosamine

Cas Number:

7512-17-6

Molecular formula:

C8H15NO6

IUPAC Name:

N-acetyl-β-D-glucosamine

Test material form:

solid

Specific details on test material used for the study:

Analysed purity: 99.42%
White powder
pH: 6.75
Batch no: RD/NAG/19/E-006
Stored at 4º C in a tight, light-resistance container

In chemico test system

Details on the study design:

Cysteine and lysine containing peptides were incubated with a positive control and the test item for 24 ± 2 hours at 22.5-30 ºC (in dark), separately.
Relative peptide concentration was measured by the high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers

Results and discussion

Positive control results:

Value of the mean percent peptide deletion of the positive control was 73% for cysteine peptide adn 49% for lysine peptide. Relative Coefficient of Variability (RCV) for positie control replicate was 0.43% for percent cysteine depletion and 1.45% for percent lysine depletion.

In vitro / in chemico

Results

Key result

Parameter:

other: Negative

Value:

1

Positive controls validity:

valid

Other effects / acceptance of results:

A standard calibration curve was generated on each day of HPLC analysis and value of r2 obtained was 0.99999 for Cysteine and 0.99998 for Lysine containing peptides (TABLE 1). This showed that system was suitable for assay.

Any other information on results incl. tables

For a valid experiment, the concentration of Cysteine or Lysine peptide was determined in each sample at 220 nm, by measuring the peak area of the appropriate peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standard. The percent peptide depletion was determined in each sample by measuring the peak area of the reference control C according to the formula described below:

Percent Peptide Depletion =    [ 1- (Peptide oeak area in replicate injection / Mean peptide peak area in reference controls C)] x 100

Cysteine 1:10/Lysine 1:50 prediction model:

Mean of Cysteine and Lysine % Depletion	Reactivity Class	Prediction
0% ≤ Mean % depletion ≤ 6.38%	Minimal Reactivity	Negative
6.38%<Mean % depletion ≤ 22.62%	Low Reactivity	Positive
22.62%<Mean % depletion ≤ 42.47%	Moderate Reactivity	Positive
42.47%<Mean % depletion ≤ 100%	High Reactivity	Positive

 

Cysteine 1:10 Prediction Model:

 

Cysteine (Cys) % Depletion	Reactivity Class	Prediction
0%≤Mean % depletion≤13.89%	Minimal Reactivity	Negative
13.89% < Mean % depletion≤23.09%	Low Reactivity	Positive
23.09% < Mean % depletion≤98.24%	Moderate Reactivity	Positive
98.24% < Mean % depletion≤100%	High Reactivity	Positive

   

 

Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers

Test Item Name	Actual % Depletion (Cysteine)	Actual % Depletion (Lysine)	% Mean Depletion (Cysteine and Lysine)	Maximum Standard Deviation (Cysteine)	Maximum Standard Deviation(Lysine)	DPRA Prediction
N-Acetyl-D-Glucosamine	1	1	1	1.05	0.44	Negative

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

From results of this study, under the specified experimental conditions, N-Acetyl-D-Glucosamine was negative in the DPRA assay.