Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January to 2 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 01-03, 2014/ signed on September 15, 2014)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: White crystalline powder
- Storage condition of test material: Room temperature

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 me thod 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-004
- Production date: not reported
- Shipping date: 27 January 2015
- Delivery date: 27 January 2015
- Expiry date: 2 February 2015
- Date of initiation of testing: 27 January 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: Number of steps not reported. Each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS 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: BMG Fluostar Optima – plate reader
- Wavelength: 540 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: mean 0.959 ± 0.019 (mean historical OD of the negative control was 0.788 ± 0.082)
- Barrier function: IC50 ≥ 1.8 mg/ml (threshold value) with 95% probability
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: For the previous 57 experiments conducted between October 2008 and December 2014 using this test method, the mean OD of the positive control was 0.168 ± 0.076 and the mean percentage viability was 21.5 ± 9.6 (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 0.788 ± 0.082 (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
- 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 ± 2 mg
- 10 ± 2 mg of the test substance was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 μL of purified water prior to application of the test substance.
- Concentration (if solution): undiluted

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:
82.6
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 (82.6 ± 4.1%)

Any other information on results incl. tables

Possible reduction of MTT by test substance

There was no change in the test substance /MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2 °C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

 

Check for colouring potential of test substance

The test substance/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

 

Table 7.3.1/1: EpiSkinresults

 

Sample

Tissue viability as percentage of mean OD negative control

Prediction
MTT endpoint

Replicate Tissues

Mean ±
SD

a

b

c

Negative Control

98.9

99.4

101.7

100.0 ± 1.5

Not applicable

Positive Control

10.1

12.1

11.6

11.3 ± 1.0

Irritant

Test item

86.1

78.1

83.6

82.6 ± 4.1

Non-irritant

 

Assay validity

Negative control: The mean absorbance of the triplicate negative control values was 0.959 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 1.5 which was below the maximum value of 18.

Positive control: The percentage mean viability of the positive control was 11.3 ± 1.0 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

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, the test substance was applied to EpiSkinhuman epidermis skin constructs (triplicate tissues). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item. The constructs were treated with the neat test substance for 15 minutes. After rinsing of the test substance the constructs were incubated for 42 hours. 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.

 

It was concluded that the test substance with a mean tissue viability of 82.6 ± 4.1%, was predicted as non-irritant to the skin.

 

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