Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-20 until 2013-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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: COMMISSION REGULATION (EU) No 640/2012 B.46. (2012)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: Yellowish crystals
- Purity test date: 19 September 2013
- Expiration date of the lot/batch: End September 2015
- Stability under test conditions: Stable until end September 2015
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

Test animals

Species:
human
Details on test animals and environmental conditions:
HUMAN SKIN: EPISKIN® (SM) (Manufacturer: SkinEthic, France, Batch No.: 13-EKIN-041, Expiry Date: 25 November 2013) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Test system

Controls:
other: Not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

Duration of treatment / exposure:
15 mins
Details on study design:
Disks of EPISKIN (3 units/chemical) were treated with the test item and incubated for 15 mins at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Mean cell viability
Value:
138
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: non irritant. (migrated information)

In vivo

Irritant / corrosive response data:
Based on the OD value the viability was 138±11.50% for the test item treated skin.
The mean OD value of the three negative control tissues was 0.800. The positive control result showed a mean of 14% viability. Each standard deviation value (SD) of the % viability was below 18. Control OD values were below historically established boundaries. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.016, Non Specific Colour % was calculated as 2.0%. Therefore additional data calculation was not necessary.
No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated % viability of the cells

Substance

Optical density

Viability (%)

Negative control (PBS)

1

0.819

102

2

0.837

105

3

0.743

93

mean ± SD

0.800

100±6.24

Positive control (5% SDS)

1

0.114

14

2

0.111

14

3

0.104

13

mean ± SD

0.110

14±0.58

Test item

1

1.101

138

2

1.197

150

3

1.012

127

mean ± SD

1.103

138±11.50

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Non Irritant (No Category).
Executive summary:

The reconstructed human epidermis model EPISKIN® (SM) is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

Disks of EPISKIN (3 units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with phosphate buffered saline (PBS). Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with the test item, the mean cell viability was 138% compared to the negative control and, therefore, non-irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid. In this in vitro EPISKIN®(SM) model test, the results indicate that the test item is Non Irritant (No Category).