Methyl α-D-mannopyranoside

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-03 to 2021-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

31 July 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl α-D-mannopyranoside

EC Number:

210-502-3

EC Name:

Methyl α-D-mannopyranoside

Cas Number:

617-04-9

Molecular formula:

C7H14O6

IUPAC Name:

(2R,3S,4S,5S,6S)-2-(hydroxymethyl)-6-methoxyoxane-3,4,5-triol

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number: 21-RHE-007
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Expiry date: 2021-02-08
- Date of initiation of testing: 2021-02-03

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: min. 25 mL DPBS, 1x
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL stock solution, 1 mg/mL in assay
- Incubation time: 3 h +/- 5 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.2 (CV=1.6 %), Acceptance criteria: OD > 0.7
- Barrier function: 7.1 h, Acceptance criteria: 4.0 h <= ET50 <= 10 h
- Morphology: Number of cell layers: 6.5, Acceptance criteria: >= 4; Satisfactory for multi-layered, highly differentiated epidermis consisting of basal, spinous and granular layers, and multi-layer stratum corneum
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Evaluation of colour interference: Test item mixed with 300 µL deionised water was incubated for 42 minutes at room temperature. Test item mixed with 1500 µL isopropanol was incubated for 120 minutes at room temperature. Since the test item has no colorant properties, no additional control was required.
Evaluation of direct reduction of MTT: Test item was added to MTT (1 mg/mL) and incubated in the dark at 37 °C for 3 h +/- 5 min. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer and no additional control was required.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive/irritant to skin if the viability is less than or equal to 50 %.
- The test substance is considered to be non-corrosive/irritant to skin if the viability greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 +/- 2 mg (to moistened skin)

NEGATIVE CONTROL
- Amount applied: 16 +/- 0.5 µL

POSITIVE CONTROL
- Amount applied: 16 +/- 0.5 µL
- Concentration: 5 % aq.

Duration of treatment / exposure:

42 +/- 1 min

Duration of post-treatment incubation (if applicable):

42 +/- 1 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

99.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

1.4

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: Test item is not a direct MTT reducer.
- Colour interference with MTT: Test item has no colorant properties

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, for details please refer to Any other information on results.
- Acceptance criteria met for positive control: Yes, for details please refer to Any other information on results.
- Acceptance criteria met for variability between replicate measurements: Yes, for details please refer to Any other information on results.
- Range of historical values if different from the ones specified in the test guideline: not applicable

Any other information on results incl. tables

Table 1: Study results:

Substance	Optical Density (OD)		Viability (%)
Negative Control 1x PBS	1	1.718	94.7
	2	1.881	103.6
	3	1.846	101.7
	mean	1.815	100
	standard deviation (SD)		4.7
Positive Control SDS (5 % aq.)	1	0.026	1.4
	2	0.024	1.3
	3	0.025	1.4
	mean	0.025	1.4
	standard deviation (SD)		7.1
Test item	1	1.812	99.8
	2	1.747	96.3
	3	1.836	101.2
	mean	1.798	99.1
	standard deviation (SD)		2.5

 

Table 2: Acceptability if the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

	Acceptance Criterion	Result
Negative control OD	≥ 0.8 and ≤ 3.0	1.718 to 1.881

 

Table 3: Acceptability of the Positive and Negative control stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.2	1.815
Mean viability positive control	< 40 %	1.4 %
SD of group-mean value	≤ 18 %	7.1 % (positive control) 4.7 % (negative control)

 

Table 4: Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.419	1.815
Mean viability positive control	≤ 2.72 %	1.4 %

 

Table 5: Test Item Data Acceptance Criteria:

	Acceptance Criterion	Result
SD of group-mean value	≤ 18 %	2.5 %

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin irritation potential.

Executive summary:

The objective of the study conducted according to OECD 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan sah after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous Solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous Solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 99.1 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin.