Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-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:

26 Sep - 01 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

adopted in 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:

adopted in 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: L´Oréal Standard Operating Procedure: “EpiSkinTM SKIN IRRITATION TEST METHOD 15 min – 42 hours” – ECVAM Skin Irritation Validation Study – VALIDATION OF THE EpiSkinTM TEST METHOD 15 min – 42 hours FOR THE PREDICTION OF ACUTE SKIN IRRITATION OF CHEMICALS.

Version / remarks:

Version 1.8, adopted February 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

(BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

6-O-α-D-glucopyranosyl-D-fructose

EC Number:

237-282-1

EC Name:

6-O-α-D-glucopyranosyl-D-fructose

Cas Number:

13718-94-0

Molecular formula:

C12H22O11

IUPAC Name:

6-O-alpha-D-glucopyranosyl-D-fructose

Test material form:

other: powder

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN-SM (TM): reconstructed three-dimensional human epidermis model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Justification for test system used:

The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured on a collagen matrix for 13 days in cell culture inserts to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.
This system is recommended in international guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (TM): reconstructed three-dimensional human epidermis model (SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 12-EKIN-035
- Date of initiation of testing: 26 Sep 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with PBS to remove any residual test item; excess PBS was removed by blotting bottom with blotting paper

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min. at 37 ± 1 °C, 5.0% CO2
- Spectrophotometer: plate spectrophotometer (Tecan Infinite 200)
- Wavelength: 550 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiSkin tissue was assessed by undertaking an MTT cell viability test with SDS. The SDS concentration to inhibit tissue viability by 50% (IC 50) was determined to be 2.2 mg/mL (acceptance criteria: ≥ 1.5 mg/mL).
- Morphology: Histology scoring of HES stained vertical paraffin sections (n = 6) was used to assess morphology of the tissues. The tissues consisited of well-differentiated epidermis consissting of a basal layer, several spinous and granular layers and a thick stratum corneum with ascore of 21.8 ± 0.3, CV = 1.3% (acceptance criteria: ≥ 19.5)
- Contamination: The cells used to produce the EpSkin tissue were screened for the presence of bacteria, fungus and mycoplasma.


NUMBER OF REPLICATE TISSUES: 3 each, for the test substance, positive and negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 15 minutes exposure and 42 h post-incubation is less or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg ± 5 µL aqua dest. (26.3 mg/cm²)

VEHICLE
5 µL distilled water (aqua dest.) were applied by a pipette to the epidermal surface in order to improve further contact between the test subsbatnce and the epidermis. The water was gently spread with the pipette. Afterwards, the tes substance powder was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount applied: 10 µL PBS

POSITIVE CONTROL
- Amount applied: 10 µL SDS
- Concentration: 5%

Duration of treatment / exposure:

15 ± 0.5 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

Remarks:

mean value of 3 tissues

Run / experiment:

15 min

Value:

95

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:
- Direct-MTT reduction: The mixture of 10 mg test item with 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
- Colour interference with MTT: The mixture of 10 mg of the test item per with 90 µL aqua dest. showed no colouring detectable by unaided eye-assessment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD550 nm of the three negative control tissues is ≥ 0.6 and ≤ 1.5
- Acceptance criteria met for positive control: mean relative tissue viability of the three positive control tissues is ≤ 40%
- Acceptance criteria met for variability between replicate measurements: the standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%

Any other information on results incl. tables

Table 2. MTT assay after 15 min exposure

	Negative Control			Positive Control			Test Item			Blank
Tissue	1	2	3	1	2	3	1	2	3
OD550	0.901	0.827	0.707	0.113	0.092	0.092	0.742	0.816	0.757	0.043
	0.899	0.817	0.697	0.107	0.088	0.087	0.741	0.813	0.762
OD550(mean, blank corrected)	0.857	0.779	0.659	0.067	0.047	0.047	0.698	0.771	0.717
SDOD550	0.089			0.011			0.034
OD550(mean values of triplicates)	0.765			0.053			0.729
SD tissue viability (%)	13.1			1.5			4.9
Viability (%)	100			7			95

According to the Test Acceptance Criteria, the validity criteria are fulfilled:

1.   mean OD550 nm of the 3 negative control tissues is ≥ 0.6 and ≤ 1.5

2.   OD₅₅₀of the blank is < 0.1

3.   mean relative tissue viability of the 3 positive control tissues is ≤ 40%

4.   the standard deviation (SD) obtained from the three concurrently tested tissues is

< 18%.

 

Applicant's summary and conclusion

Interpretation of results:

other: non-irritant according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified
Under the conditions of the reconstructed human epidermis test the test item does not possess iritant properties.