Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-MAR-2012 to 10-AUG-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Cl H-Galden (n=1)
- Physical state: colourless liquid
Specific details on test material used for the study:
- Lot/batch No.: SETTEMBRE 2011
- Expiration date of the lot/batch: 31-DEC-2016
- Storage condition of test material: ambient condition

- Purity = 74.152% w

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: N/A
Details on animal used as source of test system:
N/A
Justification for test system used:
The system is described in OECD TG No. 439
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM: Reconstructed human epidermal tissue (0.38 cm²).
- Source: SkinEthic Laboratories (France)
- Batch: 12-EKIN-024
- Examination before experiments: temperature indicator: pale grey (suitable for use); pH indicator: orange (suitable for use)

CULTURE
The test system was placed in a 12-well plate (supplied) in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium. Culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

PRELIMINARY TEST
- Non-specific reduction of MTT was evaluated as follows:
Two mL of MTT Ready-to-use Solution was incubated with 10 microliters of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

- Colouring potential test:
Chemicals’ colouring potential was assessed for potential interaction with the test system.
10 microL of the test item was added to 90 microL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes.
Colouring of the solution at the end of the incubation time was evaluated.

MAIN ASSAY
A Main Assay was carried out including the test item, positive and negative controls ( 3 replicates, each).

Exposure period:
An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature.
Adding was carried out staggering samples of approximately 1 minute.
Positive control: an intermediate re-spreading was carried out approximately after 7 minutes of incubation.

Washing:
At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

Post-exposure period:
A 42 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

Media collection for further analysis:
At the end of the incubation period, the plates were shaked on a plate shaker for about 15 minutes at approximately 300 rpm/min.
A volume of 1.6 mL of each incubation medium was removed and stored at -20°C for possible future analysis. No analysis was carried out on these samples, in agreement with the Sponsor.

MTT Assay:
The tissue insert and controls were incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity.
At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 microL of acidic isopropanol.
Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction.
At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 microL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 microL) of acidic isopropanol were analysed and used as blank.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Number of replicates:
3

Test system

Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied, concentration: 20 µL/epidermis unit each measuring 0.38 cm² (treatment level: 53 µL/cm²)
Duration of treatment / exposure:
15 ± 0.5-min exposure period
Observation period:
Following the 15-min exposure period, the tissues were washed with phosphate buffered saline (PBS). Then all tissues were incubated in SkinEthic maintenance medium for 42 hrs at 37°C, 5% CO2 and saturated humidity.
Number of animals:
None: in vitro method
Details on study design:
REMOVAL OF TEST MATERIAL
- Washing: at the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.
- Time after start of exposure: 15 min

REMARKS: the test item was applied as supplied in three replicates. Tissues were exposed to the test item in a ventilated cabinet at room temperature. Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate (SDS) solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit.

SCORING SYSTEM: the tissue insert and controls were incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hrs at 37°C, 5% CO2 and saturated humidity. At the end of the 42-hr incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank.

ACCEPTABILITY CRITERIA
Acceptability criteria were determined following set-up and validation studies.
- EPISKIN™ batch acceptance:
* Negative controls: OD values of samples ≥ 0.600, CV% ≤ 18.
* Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and CV% ≤ 18.
- Test item data acceptance: CV% ≤ 18.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test
Value:
92.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
CV% = 5.5

Any other information on results incl. tables

Table 3: Results of the main test

 

Optical density

(OD)595

Mean ±

standard deviation (SD)

CV%

OD-blank

Mean ±

standard deviation (SD)

CV%

% of viability*

Blank

0.048

0.054

0.053

0.054

0.049

0.047

0.051 ± 0.003

5.9

-

-

-

-

Negative control

0.831

0.820

0.868

0.920

0.829

0.882

0.858 ± 0.039

4.6

0.780

0.769

0.817

0.869

0.778

0.831

0.807 ± 0.039

4.8

100%

Positive control

0.085

0.065

0.070

0.064

0.084

0.074

0.074 ± 0.009

12.2

0.034

0.014

0.019

0.013

0.033

0.023

0.023 ± 0.009

39.1

2.9

Test item

0.739

0.767

0.817

0.852

0.832

0.768

0.796 ± 0.044

5.5

0.688

0.716

0.766

0.801

0.781

0.717

0.745 ± 0.044

5.9

92.3

*: referred to the negative control mean value.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the established criteria for positive results (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.
Executive summary:

The potential of the test item SOLVENT N1 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKINTM. The study was conducted according to OECD guideline 439 and in compliance with good laboratory practices (GLP).

 

The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period. The final endpoint of the assay was the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

 

Prior to the main assay, a preliminary test was carried out to assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of colouring water per se.

No interaction was recorded between the test item and MTT in test conditions similar to those of the main assay. Moreover, no colouring potential of the test item in contact with water was recorded. Thus, no additional control was added in the main phase for the evaluation of non-specific coloration which may influence evaluation of results.

 

In the main assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit each measuring 0.38 cm² (treatment level: 53 µL/cm²).

Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit.

 

The negative control gave the expected baseline value and variability, in agreement with the guideline indications. According to the method, the mean value was considered the baseline value of the experiment and thus represented 100% of cell viability.

The positive control caused the expected cell death (2.9% of cell viability when compared to the negative control) with the expected variability (CV% = 12.2).

Therefore, the assay was regarded as valid.

The test item did not induce cell death, with a mean cell viability of 92.3% when compared to the negative control. Good homogeneity was observed between replicates (CV% = 5.5).

The test item result was therefore accepted as valid.

 

According to the established criteria for positive results (cell viability less than 50%), the test item was considered to have no irritant effect on the skin under the reported experimental conditions. Therefore, the test material does not meet the classification criteria of EC Regulation No. 1272/2008 (CLP / EU GHS) and UN GHS for skin irritation.