Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2010 – 21 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with GLP and in accordance with OECD 439 Guideline for the Testing of Chemicals, adopted 22 July 2010.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

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:
other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model (2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study report included a current certificate of GLP compliance for the test facility, issued by the MHRA.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Off-white solid
- Storage condition of test material: aprox. -20 degrees in the dark.

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 0.38 cm2. The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability is 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 (OECD439) uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Test system

Vehicle:
other: Water - see comment below.
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10±2 mg of test material was dispensed over each tissue using glass weighing boats and spread over the surface of the tissue with a curved spatula. The tissues were wetted with 5 µL of distilled water prior to application of the test substance.



Duration of treatment / exposure:
15 minutes
Observation period:
Each insert (tissue) was incubated for 42±1 hours at 37±2°C in a humidified atmosphere after exposure and rinsing but prior to analysis (incubation with MTT).
Number of animals:
Triplicate tissues each for test substance, negative control (sterile Dulbecco's Phosphate Buffered Saline (DPBS) with magnesium and calcium, 10 µL), and positive control (5% Sodium Dodecyl Sulphate (SDS) in purified water, 10 µL).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Remarks on result:
other:
Remarks:
Time point: 3 hours . Remarks: The test substance has a mean tissue viability of 116.4 ± 10.4% and is predicted as a non-irritant to the skin. . (migrated information)

In vivo

Irritant / corrosive response data:
The mean value for tissue viability found for the test substance was 116.4% with a standard deviation of 10.4. On the basis that the viability value was
greater than 50%, this results therefore predicts a non irritant outcome for this endpoint.
Other effects:
Reduction of MTT by test substance: As the test substance had reduced the MTT water killed tissues (which had no metabolic activity but absorb and bind the test substance like viable tissues) were incluede in the assay as a control, together with the live tissues.

Any other information on results incl. tables

Assessment of Results

Assessment of viability

The mean Optical Density (OD) for the 6 replicate blanks was subtracted from the individual substance and control tissues OD.

The mean OD of the triplicate untreated killed tissues was subtracted from the mean OD of the triplicate killed tissues treated with the test substance to give the OD value of the MTT conversion by the test substance (nonspecific reduction of MTT). This OD value was then subtracted from the OD reading from the triplicate live tissues treated with the test substance to give the true MTT reduction by the live tissues. This corrected value was expressed as a percentage of the mean negative control value of the live tissues.

 

The viability of each tissue was expressed as a percentage of the mean negative control value.

Assay acceptance criteria

Negative control

The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be ≥0.6 and ≤1.5. The Standard Deviation (SD) value of the % viability should be <18.

Positive control

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤30% of the negative control and the SD <18.

Data interpretation - Prediction model

If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification).

Results

Possible reduction of MTT by test substance

There was no change in the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2in air. However, there was a change in colour with the test substance, the test substance/MTT solution. The test substance had interacted with the MTT, therefore additional controls were used (see section 2.3).

 

Assay validity

Negative control

The mean absorbance of the triplicate negative control values was 0.737 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation of the % viability was 2.1, which was below the maximum acceptance value of 18.

Positive control

The percentage mean viability of the positive control was 25.5±20.9 of the negative control.

The percentage mean was below the maximum acceptance value of 30% viability. The SD

(20.9) was greater than the maximum acceptance value of 18. In view of the fact that the mean percentage viability of the positive control (25.5%) was below the maximum criteria of

30%, two of the positive control replicates were very close (13.1% and 13.8%) and the test substance is clearly non-irritant and all other criteria for determination of a valid test were fulfilled, it was not considered necessary to repeat the test.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Prediction based on EPISKIN model Criteria used for interpretation of results: EU
Conclusions:
It was concluded that the test substance with a mean tissue viability of 116.4 ± 10.4%, was predicted as a non irritant to the skin.
Executive summary:

An EPISKIN skin irritation test was conducted by Huntingdon Life Sciences, UK, to assess the potential of the test substance to cause skin irritation, by means of an in-vitro test. The EPISKIN test has been accepted as a replacement to the in vivo test (Draize Skin Irritation Test), by the European Centre for the Validation of Alternative Methods (ECVAM), and an OECD Guideline 439 (2010) was followed; the study was also conducted in accordance with GLP. The test substance was applied to EPISKIN human epidermis skin constructs which 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 of the multi layers was determined by mitochondrial dehydrogenase activity. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test substance elicited a mean tissue viability of 116.4% and it was concluded that the test substance is predicted as a non irritant to the skin.