Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is comparable to a guideline study with adaptions that do not impair the overall conclusion from data. The applied tissue model and test protocol was tested in a catch-up validation study and validation results are currently asessed at EURL-ECVAM with the aim of official regulatory acceptance. Accordingly, the study follows the prediction model as described in the EURL-ECVAM "Performance Standards for in vitro Skin Irritation Testing" (ECVAM 2009). The study was carried out with reference to selected rules of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Study was performed with the Henkel OS-REp (Open Source Reconstructed Epidermis Model) model according to the Standard Operation Procedure “Catch-up Validation Study Skin Irritation: Open Source Epidermal Model; Version 3.2; revised October 2011”.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
2-Phenylethyl cyanoacrylate, Batch 3635-59, > 98.8 %

Test animals

Species:
other: Human Skin Model
Strain:
other: OS-REp reconstituted epidermis model

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Remarks:
moistured with PBS
Controls:
other: not applicable
Amount / concentration applied:
~31 µg/cm² (substabce was applied with a 25 µl spoon for the sake of practicability, see details on study design)
Duration of treatment / exposure:
0,5 h
Observation period:
42 h
Number of animals:
0
Details on study design:
Test substance:
For application of the test substance, a sharp application spoon with a volume of 25 µl was used. The spoon was filled and excess material gently scratched away without applying any pressure. The material was distributed evenly over the surface of the skin models and moistured with 25 µl PBS. The application time started after applying PBS.

Negative control:
Phosphate buffered saline, pH 7.0-7.4, with Ca/Mg (D-PBS). 25 µl of the negative control were directly dispensed atop the tissue surface. Based on historical data, the mean OD at 590 nm in the MTT assay with models treated with PBS should be located between 0.8 and 1.5 relative units.

Positive control:
5% (aq) sodium dodecyl sulphate (CAS 151-21-3). 25 µl of the postive control were directly dispensed atop the tissue surface. Viability of skin models treated with the positive control should be below 10 % based on historical data.

Treatment and MTT assay:
At the day of substance testing the OS-REp models were transferred from petri dishes to the wells of 6-well plates, where they were provided with 1 ml of fresh prewarmed culture medium each. The models were then topically exposed to the chemicals for 35 minutes each. Three tissues were used for the 2-phenylethyl cyanoacrylate, positive control (PC) and negative control (NC). After the indicated period of time the tissues were thoroughly washed with buffer solution and transferred to fresh prewarmed culture medium. After 42 hours of incubation at 37°C the tissues were transferred to 24 well-plates containing 0.2 ml of a freshly prepared MTT solution (1 mg/ml), where they remained for 3 hours. Then the blue formazan dye, which has been deposited inside living cells, was extracted with 2 ml of 2-propanol each for 2 hours at ambient temperature. The optical density of the extracted formazan solution was determined in a spectrophotometer at a wavelength 570 nm without reference wavelenght. The relative cell viability was calculated for each tissue as the percentage of the mean negative control tissues.

Data evaluation and statistical procedure:
According to the GHS classification the irritation potential for a test substance is predicted if the mean relative tissue viability of three individual tissue exposed to said substance is reduced below or equal to the 50% threshold of the mean viability of the negative controls.

In vitro result: In vivo prediction:
Mean tissue viability ≤ 50% of NC Irritant
Mean tissue viability > 50% of NC Non-irritant

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
ca. 97
Remarks on result:
other:
Remarks:
Basis: other: not applicable. Time point: 42 h. Max. score: 100.0. Reversibility: other: not applicable. Remarks: score is tissue viability as percentage of negative control. (migrated information)

Any other information on results incl. tables

All OS-REp models treated with the test substance and the controls could be included into the data analysis process. The table depicts the relative viabilities of the OS-REp models topically treated with 2 -phenylethyl cyanoacrylate and the controls. The test substance did not significantly decrease the relative tissue viability. With 97.3% the mean value is similar to that of the negative control. According to the prediction model 2 -phenylethyl cyanoacrylate can be regarded as non-irritating to the skin.

Table: Mean relative viability data of the OS-REp models after topical application of the indicated substances (mean ± SD). Viability has been determined after 35 minutes substance exposure time and 42 hours of post-incubation. All data are calculated compared to the negative control (= 100% rel. viability)

 

Test substance

Mean rel. viability

SD

Negative control

100.00

8.52

Positive control

1.33

0.47

2-Phenylethyl cyanoacrylate

97.28

10.87

 

Quality criteria:

 

- The the mean optical density of the negative control was 1,214 +/- 0,103 absorption units.

 

- The positive control (5% SDS solution) killed the tissue models nearly completely, resulting in a remaining activity of 1.33 +/- 0.47%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the test results, 2-phenylethyl cyanoacrylate is classified as being non-irritant in the in vitro skin irritation test under the described experimental conditions.
Executive summary:

2 -Phenylethyl cyanoacrylate (PhECA) was tested for skin irritation on a human three dimensional epidermal model (OS-REp, Henkel). PhECA were applied topically with the help of a 25 µl spoon to the skin model, moistened with PBS and applied for 35 minutes before the substance was washed away from the models. Application was followed by a 42 hours incubation period, then the cytotoxic (irritancy) effect was determined. 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 to the test substance. The relative mean tissue viability obtained after treatment with PhECA was 85% compared to the negative control tissues. Since the mean relative tissue viability for PhECA was above 50% it is considered to be non irritant. The positive control (SDS) had a mean cell viability of about 1 %. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 1.2. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

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