Methyl α-D-mannopyranoside

Endpoint summary

Description of key information

The test item showed no skin irritation potential (reference 7.3.1-1).

The test item showed no eye irritating potential (reference 7.3.2-1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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 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

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

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 %

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-02 to 2021-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU method B.69 RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE) TEST METHOD FOR IDENTIFYING CHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SERIOUS EYE DAMAGE

Version / remarks:

31 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 Jun 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used, and the mycoplasma status of the cell live: The EpiOcular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. Long term antibiotic and antimycotic free culture with no contamination was applicable (result: sterile). Therefore, the QA statement from the supplier considered a pass for the sterility criterion.
- Cell line used, its source, passage number and confluence of cells used for testing: Keratinocyte strain: 4F1188; passage number: not specified; Barrier function: 14.26 min (within acceptance criteria of supplier)
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212) from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot no: 30699

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg/tissue

Controls
- Amount applied: 50 µL/tissue

Duration of treatment / exposure:

6 hours (± 15 minutes)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 minutes)

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

- RhCE tissue construct used, including batch number
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 30699
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 6 hours (± 15 minutes) at 37 °C and 5 % CO2
Post-exposure immersion: 25 minutes (± 2 minutes) at room temperature
Post-exposure incubation: 18 hours (± 15 minutes) at 37 °C and 5 % CO2

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
A 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item were added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated at 37 ± 1°C and 5% CO2 and protected from light for 3 hours (± 5 minutes). Sterile deionised water (50 µL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1°C and 5 % CO2 protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties. Therefore, no additional controls were used.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled)
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues were used in this study.

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study.
Spectrophotometer used: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany

- Description of the method used to quantify MTT formazan
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (±10 minutes) at 37 °C and 5 % CO2. The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60 %. In this case no further testing in other test methods is required.
If the mean percent tissue viability is less than or equal 60 %, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Acceptable variability between tissue replicates for positive and negative controls
The results are acceptable if:
1. The negative control OD >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50 % of control viability
b) 6-hour exposure (treatment of solid test items): below 50 % of control viability

- Acceptable variability between tissue replicates for the test chemical
The difference of viability between the two relating tissues of a single chemical is <20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

- Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is two standard deviations above the current historical mean.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

89.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

30.5

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is >0.8 and <2.5 (1.435 and 1.395). For laboratory: Acceptance Criterion of mean OD ≥ 1.113, Result 1.415
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50 % of the negative control viability (30.5 %). For laboratory: Acceptance Criterion of mean viability ≤ 50.1 %, Result 30.5 %
- Other: The difference of viability between the two relating tissues of a single chemical is <20 % (values between 0.4 % to 3.9 %) in the same run (for positive and negative control tissues and tissues of single chemicals).

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.435	101.4%	1.395	98.6%	1.415	100.0%	1.98	2.8%
Positive Control	0.434	30.7%	0.429	30.3%	0.432	30.5%	0.28	0.4%
Test item	1.242	87.8%	1.297	91.7%	1.270	89.8%	2.76	3.9%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show an eye irritating potential.

Executive summary:

A study according to OECD TG 492 was conducted to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) 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 salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. All acceptability criteria after treatment with the negative control (sterile deionized water) and the positive control (methyl acetate) were met. Following treatment with the test item, the tissue viability was 89.8 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category). Under the conditions of the present study, the test item did not show an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation in vitro (reference 7.3.1-1)

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.

 

Eye Irritation in vitro (reference 7.3.2-1)

A study according to OECD TG 492 was conducted to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) 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 salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcularä-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. All acceptability criteria after treatment with the negative control (sterile deionized water) and the positive control (methyl acetate) were met. Following treatment with the test item, the tissue viability was 89.8 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category). Under the conditions of the present study, the test item did not show an eye irritating potential.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for skin and eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin and eye irritation under Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692.