Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-06 to 2017-05-15
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 06, 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm™
- Tissue batch number(s): EPI-200, Lot no. 25803
- Date of initiation of testing: 2017-03-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C for 35 min and room temperature for the last 25 min


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability is the MTT assay. The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD < 0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

- Barrier function: The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek Corporation determines the ET 50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET 50 must fall within a range established based on a historical database of results. ET-50 assay: 100µL 1% Triton x-100, 4 time points, n = 3, MTT assay: Acceptance criteria: ET-50 [4.77-8.72], Result: 5.36h.
- Contamination: HIV-1, Hepatitis B virus, Hepatitis C virus, Bacteria, yeast and other fungi - longterm antibiotic, antimycotic free culture: Not detected


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 3

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 non-irritant to skin if the viability after exposure is greater than or equal to 50% .
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg to cover the skin uniformly with an area of 0.63 cm². The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Dulbecco's phosphate buffered saline (D-PBS) 30µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): aqueous sodium dodecyl sulphate (SDS) 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test substance
Value:
117.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
optical density was 1.418
Positive controls validity:
valid
Remarks:
viability 6.1% of the negative control
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.418.

- Acceptance criteria met for positive control: The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 6.1% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.

Any other information on results incl. tables

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Table 1: Summary of results

Group

Absorbance 570 nm Tissue 1

Absorbance 570 nm Tissue 2

Absorbance 570 nm Tissue 3

Mean

Absorbance

of 3 Tissues

Relative

Standard

Deviation

[%]

Rel. Absorbance

[% of

Negative

Control]

Negative control

1.503

1.319

1.431

1.418

6.6

100.0

Positive control

0.092

0.083

0.085

0.087

5.3

6.1

Test item

1.557

1.624

1.807

1.663

7.8

117.2

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a study with alpha methyl glucoside conducted according to OECD guideline 439, the skin irritation was determined in vitro using the reconstructed human epidermis model EPIDERM ™. The cell viability was determined using the MTT assay. Since the cell viability was 117.2 % in this test and all validity criteria were fulfiled alpha methyl glucoside is considered to be non-irritating to the skin.
Executive summary:

In a study with alpha methyl glucoside conducted according to OECD guideline 439, the skin irritation was determined in vitro using the reconstructed human epidermis model EPIDERM ™. The three-dimensional human epidermis model tissue was exposed to 25 mg of the test substance on a surface area of 0.63cm² in triplicates for 60 min and subsequently washed and incubated for further 42h. 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 (deionised water) 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 60 minutes treatment with alpha methyl glucoside compared to the negative control tissues was 117.2 %. Since the mean relative tissue viability for the test substance was above 50%, alpha methyl glucoside is identified to be not irritating.