Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2011 to 21 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TCD

- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: Organic
- Physical state: Clear Liquid
- Analytical purity: ca 100%


- Lot/batch No.: M0904
- Expiration date of the lot/batch: 31 December 2013



- Storage condition of test material: Refigerated in the dark

In vitro test system

Test system:
human skin model

Test animals

Species:
other: EPISKIN three-dimensional human skin model
Details on test animals and environmental conditions:
The test involves the application of the test substance for 15 minutes to the EPISKIN three dimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

Test system

Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ?l of the test substance was dispersed over each tissue using a positive displacement pipette
Observation period:
Each insert was incubated for 42±1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. After 42 ± 1 hour each insert was transferred to a well containing 2 ml of 0.3 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
Number of animals:
Triplicate tissues each for test substance, negative control (sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium) and positive control (5% Sodium Dodecyl Sulphate (SDS) in distilled water)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
36.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
There was no change in the test substance, TCD/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.
The test substance, TCD/water solution and water control were colourless after the 15 minute shaking period. The test substance, TCD, had not shown any potential for colouring water.
The pH of the test substance, when diluted to 10% v/v with distilled water, was approximately 7.0 when measured using pH indicator paper.

Any other information on results incl. tables

Negative Control

The mean absorbance of the triplicate negative control values was 0.811 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 2.6 which was below the maximum value of 18.

Positive control

The percentage mean viability of the positive control was 11.7 ± 6.4 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:
Category 2 (irritant)
Conclusions:
It was concluded that the test substance, TCD, with a mean tissue viability of 36.1± 15.4%, was predicted as irritant to the skin.