Pentyl D-glucoside

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 14 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, Medicines & Healthcare products Regulatory Agency

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl D-glucoside

EC Number:

309-363-2

EC Name:

Pentyl D-glucoside

Cas Number:

100231-63-8

Molecular formula:

(C6H10O5)nC5H12O, n - number of D-glucopyranose units

IUPAC Name:

(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-{[(2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(pentyloxy)oxan-2-yl]methoxy}oxane-3,4,5-triol; (2S,3R,4R,5S,6S)-2-(hydroxymethyl)-6-(pentyloxy)oxane-3,4,5-triol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: EPISKIN™; reconstructed three-dimensional human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST METHOD:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 min followed by a post-exposure incubation period of 42 h. The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt within the mitochondria of viable cells in the test item treated tissues relative to the negative controls.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number: 16-EKIN-045
- Delivery date: 08 November 2016
- Maintenance Medium lot number: 16-MAIN3-075
- Assay Medium lot number: 16-ESSC-048

ADAPTATION TO CELL CULTURE CONDITIONS (PRE-INCUBATION):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item (positive and negative). The tissues were incubated at 37 °C, 5% CO2 in air overnight.

TREATMENT AND RINSING:
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with 10 µL of the test item. The test item was applied topically to the corresponding tissues ensuring uniform covering. Triplicate tissues treated with 10 μL of Dulbecco’s Phosphate Buffered Saline (DPBS) served as the negative controls and triplicate tissues treated with 10 μL of Sodium Dodecyl Sulphate (SDS) 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). The plates were kept at room temperature for 15 min.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by means of a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 h.

MTT LOADING, FORMAZAN EXTRACTION AND OPTICAL DENSITY MEASUREMENT.
Following the 42 h post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 min to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 h at 37 °C, 5% CO2 in air. At the end of the 3 h incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C for 72 h, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

NUMBER OF REPLICATE TISSUES: triplicates

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

PREDICTION MODEL:
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50% after a 15 min exposure period followed by a 42 h post-exposure incubation period
- The test substance is considered to be not irritating to skin if the viability is greater than 50% after a 15 min exposure period followed by a 42 h post-exposure incubation period

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

The test was performed in triplicates for each test or control group.

Test animals

Species:

other: in vitro system

Test system

Type of coverage:

other: in vitro system

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of all 3 test item tissues

Value:

90.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, as assessed in a suitable pre-experiment
- Colour interference with MTT: no, as assessed in a suitable pre-experiment
- IL-1alpha analysis: considered unnecessary as the results of the MTT test were unequivocal

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD of negative control 0.836 (criterion: OD ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: yes, tissue viability 11.8% (criterion: tissue viability ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: yes, SD 1.3% (negative control), 4.2% (positive control), 8.3% (test item) (criterion: ≤ 18%)

Any other information on results incl. tables

Table 1: Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control item	0.839	0.836	0.011	100.4	100*	1.3
	0.846			101.2
	0.824			98.6
Positive control item	0.138	0.099	0.035	16.5	11.8	4.2
	0.089			10.6
	0.070			8.4
Test item	0.752	0.756	0.070	90.0	90.4	8.3
	0.827			98.9
	0.688			82.3

OD = Optical Density

SD = Standard deviation

∗= The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered to be not irritating to skin based on an appropriate result in the human epidermis model test (OECD 439).