Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-28 to 2017-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 06, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC\/AM DB-ALM Protocol No. 131 “EpiSkin Skin irritation Test™15 min - 42 hours.
Version / remarks:
June 09, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
D-Glucose, reaction products with alcohols C16-18 (even numbered)
IUPAC Name:
D-Glucose, reaction products with alcohols C16-18 (even numbered)
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiSkin™ Kit Lot No.: 17-EKIN-006, SkinEthic Laboratories (69007 Lyon, France)
This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Date of initiation of testing: On 2017-02-07 (receipt of EpiSkin™) the tissues were transferred to 12-well plates containing 2 mL maintenance medium per well and incubated at 37°C ± 1°C, 5.0% CO2 for at least 24 h.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- washed once after the 15 min incubation step with DPBS. Excess DPBS was removed by blotting bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: 3 mg/mL MTT (Sigma; Lot No.: MKBR6576V) in PBS (Gibco; Lot No.: 1813255); MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL)
- Incubation time: 3 h
- Wavelength: 570 nm ± 30 nm
- Filter bandwidth: 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC 50 determination (SDS concentration, MTT test, n = 14): Specification ≥ 1.5 mg/mL; Result: 2.1 mg/mL
- Morphology: Histology scoring (HES stained vertical paraffin sections, n = 6): Specification ≥ 19.5; Result: 22.8 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: On blood of the same donors, the following was verified:
- the absence of HIV] and 2 antibodies
- the absence of hepatitis C antibodies
- the absence of hepatitis B antigen HBs
On epidermal cells of the same donors, the absence of bacteria, fungus and mycoplasma was verified.

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues were used in order to determine the non-specific reduction of MTT and if a non-specific colouring occurs that is calculated to be > 5% as compared to the negative control. The second step is necessary in order to avoid a possible double-correction for colour interference for test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues.
- N. of replicates :
Three killed tissues were incubated either with 10 mg of the test item and with the negative control (DPBS; KU), respectively, and the reduction was measured in repeat determinations.
- Method of calculation used:
If the mixture of living tissues treated with the test item and the MTT-incubation medium turns blue/purple the non-specific reduction of MTT (NSMTT) is calculated using the following formula:
NSMTT [%] = [(ODKT- QDKU)/ODNK] * 100
With KT = treated tissues; KU = negative control and NK = negative control of the living tissues.
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected according to the following formula:
TODTT = ODTM - (ODKT - ODKU)
The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:
NSCliving [%] = [ODTVT/ODUVT]*1OO
If NSCliving was ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.
If NSCliving was > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = QDTM — QDTVT

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “Category 2”, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%.
- The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is higher than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg (26.3 mg/cm²)

VEHICLE
- Amount(s) applied (volume or weight with unit): 5µL aqua dest.

NEGATIVE CONTROL
- Concentration (if solution): DPBS (Lot No.: 1737107)

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulfate (SDS; Lot No.: 40015277) in aqua dest.
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
three tissues were used and the viability measured in repeat determinations

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
106.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
mean OD570 = 0.644
Positive controls validity:
valid
Remarks:
mean tissue viability = 15.5%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: Yes, ≤ 5%


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
OD 570 ± 30 nm blank: mean: 0.043; SD: 0.001; n: 67
Absolute OD 570 ± 30 nm NK: mean: 0.866; SD: 0.120; n: 66
Relative Viability [%] PC: mean: 11.73; SD: 8.17; n: 67
SD of Viability [%]: mean: 6.43; SD: 4.68; n: 254

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
D-Glucose reaction products with alcohols C16-C18 was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), D-Glucose reaction products with alcohols C16-C18 was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with DPBS to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37 ± 1°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (aqua dest.) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment and 42 h post-incubation with D-Glucose reaction products with alcohols C16-C18 compared to the negative control tissues was 102 %. Since the mean relative tissue viability for the test substance was above 50%, the test item is identified to be not irritating.