4-O-α-D-glucopyranosyl-D-glucitol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 August 2018 to 05 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-O-α-D-glucopyranosyl-D-glucitol

EC Number:

209-567-0

EC Name:

4-O-α-D-glucopyranosyl-D-glucitol

Cas Number:

585-88-6

Molecular formula:

C12H24O11

IUPAC Name:

4-O-α-D-glucopyranosyl-D-glucitol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: ELEL7
- Expiration date of the lot/batch: 07 October 2022

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material) : tested as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : not applicable

OTHER SPECIFICS: none

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: three-dimensional human epidermis model (EPISKINTM (SM))

Cell source:

other: adult donors

Source strain:

other: not applicable

Details on animal used as source of test system:

Not applicable (EPISKINTM (SM) kits).

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

prior to test item application, distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) model
- Tissue batch number(s): 18-EKIN-036
- Production date: 04 September 2018
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 05 September 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature (23.4-25.3°C)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: volume not specified, once (After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: none specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL (MTT working solution)
- Incubation time: 3 hours at 37°C
- Spectrophotometer: not specified
- 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
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study) and were documented in an appendix to the study report.
NUMBER OF REPLICATE TISSUES: three replicates were used for the test item

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : not applicable (the test material did not interact with MTT).

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.
-The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls.
-In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable (not different from TG 439 recommendations).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes (± 0.5 min) at room temperature (23.4-25.3°C).

Duration of post-treatment incubation (if applicable):

42 hours + 3 hours (MTT test)

Number of replicates:

3 replicates were used for the test item.
3 negative controls and 3 positive controls were also run in the assay.
Furthermore, as the test item was coloured, 2 additional test item-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative viability compared to the negative control

Run / experiment:

1 (mean three samples)

Value:

93.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none specified.
- Direct-MTT reduction: no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT.
- Colour interference with MTT: The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.3%

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS: All thee parameters below met the acceptability criteria, therefore the study was considered to be valid.
- Acceptance criteria met for negative control: As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Acceptance criteria met for positive control: As the test item was coloured (white), two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.3%. This value was below 5%, therefore additional data calculation was not necessary (mean blank value was 0.048).
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.1%.
- Range of historical values if different from the ones specified in the test guideline:

Negative control (PBS) (number of cases 251)
Mean optical density (OD): 0.788 ± 0.129 (SD) (Min. OD: 0.573 and Max. OD: 1.362)

Positive control (5% (w/v) SDS solution) (number of cases 246):
Mean optical density (OD): 0.065 ± 0.041 (SD) (Min. OD: 0.019 and Max. OD: 0.354)

Any other information on results incl. tables

Table 1 : Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (% RV)	Standard Deviation
		Measured	Blank corrected
Negative control (Phosphate buffered saline)	1	0.834	0.786	97.0	2.7
	2	0.866	0.818	100.9
	3	0.876	0.828	102.1
	mean	-	0.811	100.0
Positive Control (5% (w/v) SDS solution)	1	0.072	0.025	3.0	1.0
	2	0.076	0.029	3.5
	3	0.088	0.040	4.9
	mean	-	0.031	3.8
Test item (Maltitol)	1	0.807	0.760	93.7	3.1
	2	0.833	0.786	96.9
	3	0.783	0.736	90.7
	mean	-	0.760	93.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this in vitro skin irritation study, the test item Maltitol, is not irritant to skin. The test material is not classified as irritating to the skin according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Executive summary:

This GLP compliant study was performed to assess the skin irritation potential of the test item Maltitol in vitro using the reconstructed human epidermis model EPISKINTM (SM). This test was performed according to the OECD Test Guideline No. 439.

Material and methods

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of color contribution (NSCliving) from the test item. The test item did not react with MTT and therefore the use of additional controls was not necessary. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Results

Following exposure with Maltitol, the mean cell viability was 93.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Conclusion

In conclusion, under the experimental conditions of this in vitro skin irritation study, the test item Maltitol, is not irritant to skin. The test material is not classified as irritating to the skin according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.