Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 October 2012 to 08 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 439 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspection date: 10 July 2012/ signed on 30 November 2012)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Description: clear colourless liquid
Specific details on test material used for the study:
- Storage conditions: room temperature in the dark
- Expiration date of the lot/batch: June 2013

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 12-EKIN-036
- Production date: not reported
- Shipping date: not reported
- Delivery date: 02 October 2012
- Date of initiation of testing: 10 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. Remaining DPBS removed with absorbent paper, and if necessary with a cotton-bud.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without a reference filter
- Filter bandwidth: NA
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Histology scoring : 21.9 +/- 0.5 (within the specification, i.e. > 19.5)
- Barrier function: IC50 = 2.1 mg/mL (within the specification, i.e. > 1.5 mg/mL)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous ten months. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : not applicable

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 irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.
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
- Concentration (if solution): The test item was used as supplied (undiluted).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): 5% SDS in distilled water
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate tissues for test substance, negative and positive controls

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period
Value:
40.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.8%
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 0.644 and the standard deviation value of the percentage viability was 6.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.2%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 2.7%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Since October 2009 to the commencement of this study (124 studies) the historical mean OD540 of the positive control was 0.059 ± 0.022 and the mean percentage viability was 7.4 ± 3.2. In this same period the historical mean OD540 of the negative control was 0.797 ± 0.086. The positive and negative controls were within the historical ranges for this testing facility.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540of tissues

Mean OD540of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.630

0.644

0.041

97.8

100*

6.4

0.612

95.0

0.691

107.3

Positive Control Item

0.039

0.044

0.008

6.1

6.8

1.2

0.053

8.2

0.039

6.1

Test Item

0.243

0.262

0.017

37.7

40.7

2.7

0.269

41.8

0.275

42.7

SD=   Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test substance is classified for skin irritation Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. 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 (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) 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.

This test was designed to be compatible with the OECD Guideline No. 429 and method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density was measured at 540 nm.

The relative mean viability of the test item treated tissues was 40.7 ± 2.7 %, after the 15‑minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

With a relative mean tissue viability < 50 %, the test material was considered to be irritant.

Under the experimental conditions of this study, the test substance is classified for skin irritation Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.