D-mannose

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

D-mannose

EC Number:

222-392-4

EC Name:

D-mannose

Cas Number:

3458-28-4

Molecular formula:

C6H12O6

IUPAC Name:

D-mannose

Test material form:

solid: particulate/powder

Details on test material:

Purity: 100%

In chemico test system

Details on the study design:

The test article was incubated concurrently in two separate buffers, one with cysteine (Ac-RFAACAA-COOH) and one with lysine (Ac-RFAAKAA-COOH). Reactive chemicals bind one or both of the peptides thereby reducing their free concentration levels. The disappearance of each peptide is measured by HPLC/UV. This method does not require any biological material such as enzymes in order for this reaction to take place. It is important to note that the cysteine peptide captures soft electrophiles, while the lysine peptide captures hard electrophiles. This makes the DPRA assay a good choice to screen for reactive chemicals which are associated with allergic contact dermatitis.

D-Mannose was prepared at a stock concentration of 100 mM in water and added to the peptide reactions. 2,3-butanedione, the positive control material, was prepared in acetonitrile to yield a final concentration of 100 mM.

The cysteine peptide was prepared at 0.667 mM in Cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile or water containing no reference or test articles.
Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. Aliquots of 1 mL were added to 9 glass vials for each peptide and placed at the beginning middle and end of the samples. These reactions were used to ensure the consistency of peptide detection during the run.
A standard curve was prepared for both peptides by adding 400 µL of acetonitrile to 1600 µL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6 point standard curve. A zero peptide standard was also included in the standard curve.

After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV.(HPLC with gradient elution and UV detections at 220 nm using a Waters Alliance 2795 HPLC equipped with a 2996 Photodiode Array). Prior to sample analysis the suitability of the HPLC/UV system was verified by running the standard curve and a triplicate set of reference controls. Samples were run on an Agilent Zorbax SB-C-18 column. Sample analysis was initiated within 24 ±2 hours of the reaction start. The results were acquired with the MassLynx data system and quantified via the QuanLynx application. Samples were injected once.

After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula:
% Depletion = (1-(test compound area/ vehicle control area)) x 100

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: % CYS depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Parameter:

other: & Lys depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Parameter:

other: % DPRA depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

For this test: low, moderate, and high reactivity were considered positive, while minimal reactivity was considered negative. See results in the table below.

It should be noted that some co-elution of mannose test material with peptide was observed with the lysine peptide. The co-elution was found to be consistent across all the replicates, and consisted of small “shoulders” on the main peptide peak. The peak integration used to calculate the depletion was the one initially predicted by the computer. Modification of the peak by the analytical scientist showed that removing the “shoulder” from the main peptide peak area did not greatly change the depletion. This indicated the co-elution did not significantly increase the signal. No co-elution issues were observed with the cysteine peptide.

Material	%Cys Dep	SD	%Lys Dep	SD	%Dep DPRA	Reactivity Class	Sensitizer
2,3-butanedione	79.7	1.3	27.8	2.8	53.8	High	Positive
D-Mannose	0.0	4.6	0.0	3.1	0.0	Minimal	Negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

D-Mannose showed minimal reactivity in the DPRA test and therefore is considered to be negative for skin sensitisation.

Executive summary:

D-Mannose was tested in the OECD guideline study 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA). The overall DPRA depletion for D-Mannose was 0.0%. It should be noted that some co-elution of test material with peptide was observed with the lysine peptide reactions. The co-elution was found to be consistent across all the replicates, and consisted of small “shoulders” on the main peptide peak. The peak integration used to calculate the depletion was the one initially predicted by the computer. Modification of the peak by the analytical scientist showed that removing the “shoulders” from the main peptide peak area did not greatly change the depletion. This indicated the co-elution did not significantly increase the signal. No co-elution issues were observed with the cysteine peptide. D-Mannose was negative for sensitization potential with minimal to no reactivity with either peptide.