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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:
February 17th, 2017 to March 27th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal
Batch No.: SC00019822
Expiry date: 01 March 2019

In vitro test system

Test system:
human skin model
Remarks:
EPISKIN Small ModelTM
Source species:
human
Cell type:
other: EPISKIN-SMTM, 0.38 cm2
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Details on animal used as source of test system:
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.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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 item was applied undiluted (25 µl) directly on top of the tissue
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Duration of treatment / exposure:
CETONAL was applied undiluted (25 µl) directly on top of the skin tissue for 15 ± 0.5 minutes.
Duration of post-treatment incubation (if applicable):
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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
second test after treatment with CETONAL
Value:
33
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

The positive control had a mean cell viability of 12% 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 with the control items was less than 7%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 57% (individual values 38%, 100% and 33%). However, the standard deviation value between tissue replicates of CETONAL was 37% after 15 minutes exposure which is not within the acceptability criteria of the assay (≤18%). Therefore the test was repeated.

The relative mean tissue viability obtained in the second test after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 43% (individual values 59%, 44% and 26%).

The relative mean tissue viability obtained in the second test after treatment with CETONAL compared to the second set of negative control tissues with a standard deviation of 11% was 33% (individual values 46%, 34% and 20%).

The positive control had a mean cell viability of 7.3% after 15 ± 0.5 minutes exposure. 

Since the mean relative tissue viability for CETONAL was below 50% after 15 ± 0.5 minutes treatment in 4 out of 5 tissues, CETONAL is considered to be irritant.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, CETONAL is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate CETONAL for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of CETONAL 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 SC00019822 of CETONAL was a pale yellow liquid with a purity of 98.02%. CETONAL 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.

Skin irritation is expressed as the remaining cell viability after exposure. 

The positive control had a mean cell viability of 12% 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 with the control items was less than 7%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 57% (individual values 38%, 100% and 33%). However, the standard deviation value between tissue replicates of CETONAL was 37% after 15 minutes exposure which is not within the acceptability criteria of the assay (≤18%). Therefore the test was repeated.

The relative mean tissue viability obtained in the second test after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 43% (individual values 59%, 44% and 26%).

The relative mean tissue viability obtained in the second test after treatment with CETONAL compared to the second set of negative control tissues with a standard deviation of 11% was 33% (individual values 46%, 34% and 20%).

 The positive control had a mean cell viability of 7.3% after 15 ± 0.5 minutes exposure. 

Since the mean relative tissue viability for CETONAL was below 50% after 15 ± 0.5 minutes treatment in 4 out of 5 tissues, CETONAL is considered to be irritant.

In conclusion, CETONAL is irritant in the in vitro skin irritation test under the experimental conditions described in this report.