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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Mar 2015 to 22 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(4-isobutyl-2-methylphenyl)propanal
EC Number:
811-285-3
Cas Number:
1637294-12-2
Molecular formula:
C14H20O
IUPAC Name:
3-(4-isobutyl-2-methylphenyl)propanal
Test material form:
liquid
Specific details on test material used for the study:
Name (as stated in the report): GR-88-0778
Lot No: Batch 6
Aspect: Colourless liquid
Expiration date: March 29, 2017

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM), 0.38 cm2 , Batch no.: 15-EKIN-020)
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
not specified
Details on animal used as source of test system:
adult human-derived epidermal keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
The liquid test substance was applied undiluted (25 μl) directly on top of the tissue.
Details on test system:
- Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 15-EKIN-020)
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 μl (undiluted) directly on top of the skin tissue
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hour post-incubation period
Number of replicates:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Since the mean relative tissue viability for GR-88-0778 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.
Other effects / acceptance of results:
The positive control had a mean cell viability of 24% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Any other information on results incl. tables

GR-88-0778 was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that GR-88-0778 did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by GR-88-0778 was 3% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. The mean absorption at 570 nm measured after treatment with GR-88-0778 and controls are recorded. The individual OD570 measurements are also recorded and shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues was 32%. Since the mean relative tissue viability for GR-88-0778 was below 50% it is considered to be irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 24%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Finally, it is concluded that this test is valid and that GR-88-0778 is irritant in the in vitro skin irritation test under the experimental conditions described in the report.
Executive summary:

In vitro skin irritation test with GR-88-0778 using a human skin model.

This report describes the ability of GR-88-0778 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of GR-88-0778 was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch 6 of GR-88-0778 was a colourless to pale yellow liquid with a purity of 98.6%. GR-88-0778 was applied undiluted (25 μl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

GR-88-0778 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by GR-88-0778 was 3% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-88-0778 compared to the negative control tissues was 32%. Since the mean relative tissue viability for GR-88-0778 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant. The positive control had a mean cell viability of 24% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that GR-88-0778 is irritant in the in vitro skin irritation test under the experimental conditions described in this report.