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Description of key information

Skin irritation - in vitro

The test substance was applied to EPISKIN human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. 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. The test substance, TCD, elicited a mean tissue viability of 36.1 ± 15.4 % and was predicted as an irritant to the skin.

Eye irritation - in vitro

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance. Sodium hydroxide (10 %) was tested in parallel as a positive control. The assay used isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vitro. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which can be used to classify and rank test substances as potential eye irritants according to OECD guideline 437 (OECD 437).

The test substance elicited an In Vitro Irritancy Score of 1.5 ± 0.7 and was predicted to be a non-corrosive/non-severe eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 system:
human skin model
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.
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)
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.

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.

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October to 13 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eyes
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µl

Duration of treatment / exposure:
10 min ± 30 s
Observation period (in vivo):
n/a
Number of animals or in vitro replicates:
n/a
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure: 2 h 10 m

SCORING SYSTEM: opacitometer

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
in vitro irritation score
Value:
1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with test substance, TCD, the corneas were noted as clear. The corneas treated with the positive control, sodium hydroxide 10% (w/v), were very opaque and the corneas treated with the negative control, 0.9 % saline, were clear.

The test substance was diluted to 10 % w/v with 0.9 % saline to obtain an aqueous solution for pH measurement. The pH of the test sample, measured using universal pH sticks, was approximately 7.0.

The positive control, sodium hydroxide (10 %), elicited an In Vitro Irritancy Score of 277.1 ± 19.1. This value was within the historical range (mean ± 2 x SD, 256.5 - 314.9) for the assays performed to date.

The negative control, 0.9 % saline, opacity mean change value was 1.0 which was below the maximum acceptance value of 2.0. The permeability mean of the negative control was 0.018 which was below the maximum acceptance value of 0.1.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance, TCD, elicited an In Vitro Irritancy Score of 1.5 ± 0.7 and was predicted to be a non-corrosive/ non-severe eye irritant.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:

This was the only study available

Justification for selection of eye irritation endpoint:

This was the only study available

Effects on skin irritation/corrosion: slightly irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Skin Irritation / corrosion

According to 3.2.2.1.2.4 of the Guidance on the Application of CLP, the EPISKIN test can be used for purposes of classification. According to the test method, OECD Guideline 439, substances that show a positive response in the EPISKIN test but for which the mean tissue viability is less than 50 % are classified as GHS Category 2 which is equivalent to CLP Category 2.

The mean tissue viability for TCDV is 36.1 % and therefore this substance is classified under CLP Regulation 1272/2008 as category 2 irritant, for skin irritation without the need for any further (in vivo) testing.

Eye irritation / corrosion

According to 3.3.2.1.2.4 of the Guidance on the Application of CLP, the BCOP test can be used for purposes of classification.

According to OECD Guideline 437, the BCOP test can be used to classify substances under the GHS classification scheme as a severe eye irritant. The test can also be used to identify chemicals that do not require classifcation for eye irritation or severe eye irritation where an in vitro irritancy score is ≤3.

The in vitro irritancy score for TCD is 1.5 ± 0.7 and therefore this substance is not classifed for eye irritation or severe eye irritation under the GHS classifciation scheme, which is equivalent to being not classified under CLP Regulation 1272/2008.