Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 to 21 September 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 July 2015
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 Program (inspected on June 17, 2015 / Signed on September 24, 2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colorless liquid
Specific details on test material used for the study:
Storage condition of test material: Room temperature in the dark

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): 15-EKIN-037
- Production date: not reported
- Shipping date: 15 September 2015
- Delivery date: 15 September 2015
- Expiry date: 21 September 2015
- Date of initiation of testing: 16 September 2015

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: not reported. At the end of the treatment period, each tissue was rinsed with Dulbeccos Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ to remove any residual test or control items and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air.
- 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: 562 nm (without a reference filter)
- Filter: not appicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.722, 0.732 and 0.702 (mean historical OD of the negative control was 0.854 ± 0.148)
- Barrier function: IC50 = 1.9 mg/ml ( ≥ 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: For the previous 28 experiments conducted between 09 March 2015 and 14 September 2015 using this test method, the mean OD of the positive control was 0.091 ± 0.037 and the mean percentage viability was 10.5 ± 4.8 (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18%). In this same period the mean OD of the negative control was 0.854 ± 0.148 (The assay establishes the acceptance criterion for an acceptable test if the mean OD 562 for the negative control treated tissues was ≥0.6 and ≤1.5).

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
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL /cm2) of the test item was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
Duration of treatment / exposure:
The EpiSkin™ human epidermis skin constructs were treated with the undiluted test item for an exposure period of 15 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.
Number of replicates:
Triplicate tissues for test item, 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:
104
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:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

Any other information on results incl. tables

Direct MTT Reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

 

Assessment of Color Interference with the MTT endpoint: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

Table 7.3.1/1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item 

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

±SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.722

0.719

0.015

100.4

100*

2.1

0.732

101.8

0.702

97.6

Positive Control Item

0.085

0.082

0.006

11.8

11.4

0.9

0.075

10.4

0.087

12.1

Test Item

0.751

0.748

0.021

104.5

104.0

2.9

0.768

106.8

0.726

101.0

 

SD=Standard deviation; *= The mean viability of the negative control tissues is set at 100%; OD562= Optical Density

 

Assay validity

The relative mean tissue viability for the positive control treated tissues was 11.4% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.719 and the standard deviation value of the viability was 2.1%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.9%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test item is not classified as a skin irritant 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 principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item.  Triplicate tissues were treated with 10 μL (26.3 μL/cm2) of the undiluted test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

 

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance with a mean tissue viability of 104.0 ± 2.9 %, was predicted as non-irritant to the skin. The quality criteria required for acceptance of results in the test were satisfied.

 

Under the experimental conditions of this study, the test item is not classified as a skin irritant 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.