6-deoxy-L-mannose

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

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.

L-Rhamnose 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

Key result

Parameter:

other: % CYS depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: & Lys depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: % DPRA depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

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
L-Rhamnose	0.0	5.1	0.0	5.0	0.0	Minimal	Negative

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

L-Rhamnose was tested in the OECD guideline study 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA). The overall DPRA depletion for L-Rhamnose 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. L-Rhamnose was negative for sensitization potential with minimal to no reactivity with either peptide. 

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

KeratinoSens cells were plated on 96-well tissue culture treated black walled clear bottom polystyrene plates, 125 μL per well. The cells were left for 24 hours to adhere and the media was replaced with a low serum medium prior to dosing with the test compound at a range of 12 concentrations. The compound was dosed on two separate plates, one to determine luminsence (Keap1-Nrf2-ARE pathway) and a second to determine cytotoxicity (MTT cell viability assay). Following an incubation period of 48 hours, the cells were either lysed and assessed for the luciferase reporter gene expression using a luminescent assay (Luciferase Assay Kit, Promega) and the BioTek luminometer or loaded with MTT [yellow; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. After 4 hour incubation the resulting formazan from the MTT cell viability assay was solubilised in a 10% SDS solution. The plates were then scanned at 570 nm using a SunriseTM microplate absorbance reader (TECAN).
A compound is rated a skin sensitizer in the KeratinoSens assay if the reporter gene expression is induced above a fold of 1.5 (EC1:5) at concentrations below 1000 μM (or 200 μg/mL) with no observed reduction in cell viability of greater than 30%. Increased reporter gene expression in cells with less than 70% cellular viability (IC30) could be due to cell stress than rather than a potential skin sensitizing effect.
Incubation time: 48 h
Concentrations (μM): 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000
Replicates per concentration: 3
Cell model: KeratinoSens
Vehicle: Water
MW: 164.47
KeratinoSens vehicle SD: 19% (passed), 10% (passed), 12% (passed)

Key result

Run / experiment:

other: Mean of Exp. 1 - 3

Parameter:

other: Calc: EC1:5 (μM)

Value:

2 000

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Calc: EC1:5 was >2000 μM indicating no sensitising potential in the KeratinoSens assay

Executive summary:

The test substance was tested in an in vitro ARE-Nr f2 Luciferase skin sensitisation assay in accordance with OECD Guideline 422D at concentrations of 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 μM. The compound was dosed on two separate plates, one to determine luminsence (Keap1-Nrf2-ARE pathway) and a second to determine cytotoxicity (MTT cell viability assay). Three trials were run. Following an incubation period of 48 hours, the cells were either lysed and assessed for the luciferase reporter gene expression using a luminescent assay (Luciferase Assay Kit, Promega) and the BioTek luminometer or loaded with MTT [yellow; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. After 4 hour incubation the resulting formazan from the MTT cell viability assay was solubilised in a 10% SDS solution. The plates were then scanned at 570 nm using a SunriseTM microplate absorbance reader (TECAN). The test substance has an EC1:5 of >2000 μM indicating that it had no sensitising potential in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance was negative for skin sensitisation in two in vitro studies and did not produce a sensitisation response in humans when tested as 10% of a leave-on facial product. Therefore, no classification is required for skin sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.