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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 - 14 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 26 July 2013
GLP compliance:
yes (incl. certificate)
Remarks:
GROUPE INTERMINISTERIEL DES PRODUITS CHIMIQUES, France

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fatty acids, vegetable-oil, esters with dipropyleneglycol
- Physical state: liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: ES 110680
- Expiration date of the lot/batch: 25 October 2016
- Storage condition of test material: at room temperature

Test animals

Species:
other: reconstructed human epidermis
Strain:
other: SkinEthic, RHE/S/17 Batch No. 14 RHE 0407
Details on test animals and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic (Batch No. 14 RHE 0407)
- Adaptions to cell culture conditions: Upon receipt, the tissues were transferred into 24-well plates containing 300 µL maintenance medium per well.

ENVIRONMENTAL CONDITIONS (Incubator)
- Temperature (°C): 37 ± 2
- CO2 gas concentration (%): 5%
- Humidity: maximum

Test system

Type of coverage:
open
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: concurrent negative control tissue treated with phosphate buffered saline
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL

To ensure a good contact with the epidermis, during all the treatment period, the test item was recovered with a nylon mesh provided by SkinEthic.



Duration of treatment / exposure:
42 min exposure
41.5 h post-incubation
Observation period:
Not applicable
Number of animals:
Not applicable. Tests were performed in triplicates.
Details on study design:
TEST SITE
-Area of exposure: 0.5 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: After the incubation period, the inserts were washed with PBS (25 x 1 mL).
- Time after start of exposure: 42 min

CELL VIABILITY MEASUREMENTS
- Method: MTT assay
- Details on method used: For viability testing, the skin samples were placed in 300 µL MTT solution (1 mg/mL) for 3 h at 37 °C and 5% CO2. Afterwards, the precipitated blue formazan product was extracted in isopropanol for 2 h under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extractions in isopropanol (1:2). The absorbance was measured in triplicates of MTT extract in ELx800 absorbance microplate reader supplied by Biotek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

PRE-EXPERIMENTS
- To evaluate non-specific MTT-reducing capability of the test item, 16 µL of the test item were mixed with 300 µL MTT solution (1 mg/mL) similar to the procedure performed in the main test. No blue or purple colouration of the MTT solution was determined.

EVALUATION CRITERIA
The irritancy potential of the test item was evaluated based on relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS. The substance is considered to be irritant to skin if the tissue viability after 42 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance is considered as non-irritant to skin if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: cell viability (% of negative control)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: negative control (PBS). Time point: approx. 45 h (42 min exposure followed by 41.5 h post-incubation and 3 h incubation in MTT solution). (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (% of negative control)
Value:
84.2
Remarks on result:
other:
Remarks:
Basis: other: test material. Time point: approx. 45 h (42 min exposure followed by 41.5 h post-incubation and 3 h incubation in MTT solution). (migrated information)
Irritation / corrosion parameter:
other: other: (% of negative control)
Value:
1.1
Remarks on result:
other:
Remarks:
Basis: other: positive control (5% SDS). Time point: approx. 45 h (42 min exposure followed by 41.5 h post-incubation and 3 h incubation in MTT solution). (migrated information)

In vivo

Irritant / corrosive response data:
Application of the test item on reconstructed human epidermis for 42 minutes resulted in a cell viability of 84.2% compared to the concurrent negative control tissues.
Other effects:
No further effects were reported.

Any other information on results incl. tables

Table 1. Summary of results

Test item

Total OD570 of 3 replicate tissues

(mean of 3 extracts from 3 tissues, triplicate measurement per extract)

Mean relative tissue viabilitiy

[%]

SD

Negative Control PBS

1.156

100

1.7

Test Item

0.974

84.2

14.9

Positive Control

5% SDS

0.013

1.1

0.0

OD: Absorption at 570 nm

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test item showed no irritant effects. The test item is therefore classified as not irritating.
CLP: non classified
DSD: non classified