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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 - 14 Dec 2015
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:
2016

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:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
amended 20 Jul 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl ascorbic acid
- Storage condition of test material: in the freezer (≤ -15 °C)
- Substance type: white to very pale yellow, crystalline, powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors
Source strain:
other: EpiDerm™; reconstructed three-dimensional human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model (SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 15-EKIN-049
- Expiry date: 14 Dec 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.0 - 37.2 °C
- Temperature of post-treatment incubation: 36.0 - 37.2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The test substance was washed from the skin surface with PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the final product was assessed by undertaking an MTT cell viability and cytotoxicity test with sodium dodecyl sulphate.
- Morphology: Histological examination was performed to demonstrate a human epidermis-like structure. A well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum was observed.
- Contamination: All biological components of the epidermis were tested for the presence of viruses, bacteria, fungus and mycoplasma prior to the test.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Only a single experiment was conducted.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and a 42 h post-incubation period is less than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10.9 - 11.1 mg (skin was moistened with 5 µL Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
in triplicates for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
15 minutes exposure
Value:
92
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: The percent tissue viability is >50%, indicating the test substance will not be irritating to skin
Other effects / acceptance of results:
- Direct-MTT reduction and colour interference with MTT: The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that the test substance did not interact with the MTT endpoint.

Any other information on results incl. tables

Table 1. Analysis of tissue functionality and quality (provided by supplier)

 

Specification

Result

Histology scoring (HES stained vertical paraffin sections, n = 6)

≥ 19.5

21.9 ± 0.4 (CV = 1.7%)

IC50 determination (SDS concentration, MTT, n = 14)

≥ 1.5 mg/mL

2.0 mg/mL

 

Table 2. Historical control data.

 

Negative Control

Positive Control

 

OD570

OD570

Viability (%)

Range

0.576 - 1.352

0.020 - 0.408

2.1 - 43.0

Mean

1.03 ± 0.15

0.14 ± 0.11

14.8 ± 10.7

Control data obtained by collecting all data over the period of March 2012 until March 2015.

 

Table 3. MTT assay after 15 min exposure.

 

Negative control

Positive control

Test substance

Tissue sample

1

2

3

1

2

3

1

2

3

OD570

0.900

0.676

0.736

0.108

0.110

0.135

0.680

0.753

0.688

OD570 (mean values of replicates)

0.770 ± 0.116

0.118 ± 0.015

0.707 ± 0.040

Viability (% of control)

100

15

92

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met