Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February - 5 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted by GLP accredited laboratory. Method according to OECD guideline.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tropicate
- Substance type: pure active substance
- Physical state: liquid

Test animals

Species:
human

Test system

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
10µl undiluted test material. The same amount is applied to the positive and negative control.
Duration of treatment / exposure:
15 minutes at room temperature.
Details on study design:
Prior to the analysis, the test substance was checked for possible direct reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; (CAS number 298-93-1) (MTT).

10 µl of the test substance was added to 2.2 ml MTT solution and incubated for 3h at 37°C, 5% CO2. A negative control of distilled water was included.
Three tissues per test were treated with the test material. Three tissues were treated with the negative control and three tissues were exposed to the positive control (5% aqueous solution of sodium dodecylsulphate-SDS CAS 151-21-3). The tissue samples were exposed to the test substance / controls for 15 minutes. After the exposure, the tissues were rinsed with phosphate buffered saline (PBS) and incubated for 42h at 37°C, 5% CO2.
 
After the incubation, the tissues were subsequently incubated for another 3h in the presence of 2.2ml MTT. The formed formazan was extracted with 0.5 ml isopropanol for 2h under agitation in the dark. The amount of Formazan was determined spectrophotometrically (Optical Density - OD) at 570 nm in duplicate. The cell viability was measured against the mean of the negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
7.5
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. (migrated information)

In vivo

Irritant / corrosive response data:
The test substance was visually checked for possible direct MTT reduction causing a colour change. No interaction with MTT was observed.
Details of the data and the mean of the spectroscopic measurements can be found in Table 1. Table 2 presents the mean relative tissue viability obtained after 15 minutes treatment with the test substance being the mean of the absorption of the test substance divided by the negative control mean for each application. Since the mean relative tissue viability of the test substance was below 50% after an exposure duration of 15 minutes, the test substance is considered to be irritant.

Any other information on results incl. tables

Table 1  Absorptions (OD570) in the in vitro skin irritation test

15-minute exposure
1 2 3 Mean
Negative control 0.548 0.529 0.507 0.528
Test substance 0.029 0.050 0.040 0.040
Positive control 0.104 0.071 0.076 0.084

Table 2 Mean tissue viability as % of negative control in the in vitro skin irritation

Exposure 15-minutes SD
%
Negative control 100 3.8
Test substance 7.5 2.0
Positive control 15.9 3.4

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test is complies with the acceptability criteria and the test substance is considered an irritant in the in vitro skin irritation test under the experimental conditions used.
Executive summary:

An in vitro skin irritation test was conducted on a human skin model according to the OECD guideline 439. The basis of the test is that the cell viability was determined by using the MTT reduction assay. The test substance was applied undiluted on top of the skin tissue for 15 minutes. After the exposure, the tissue was incubated with MTT and the formazan that was produced from MTT was spectroscopically determined at 570 nm. The relative mean tissue viability of the test substance expressed as the absorption of the test substance against the negative control showed that the test substance has a relative mean tissue viability of ≤50% compared to the negative control. The test substance is therefore considered an irritant.