Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Feb 2012 - 13 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012
Reference Type:
other: Amendment to study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 2004
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): GSID 3056-1
- Physical state: solid, brownish
- Analytical purity: 98% (see amendment)
- Lot/batch No.: 1470 VB04
- Storage condition of test material: room temperature
- Stability under storage conditions over the study period: guaranteed by the sponsor (sponsor holds this responsibility)

Test animals

Species:
other: not applicable since in vitro test
Strain:
other: not applicable since in vitro test
Details on test animals and environmental conditions:
In vitro test with reconstructed human epidermis (RHE):
- The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
- The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Supplier: Tissue model Epi-200 obtained from MaTek Corp., Ashland, USA
- Assay Medium: Dulbecco's modified eagle's medium (DMEM)

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
other: water or PBS
Controls:
other: negative control: concurrent vehicle; positive control for corrosion test: 8 N potassium hydroxide; positive control for irritation test: 5 % sodium dodecyl sulfate (SDS)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL (corresponding to 13 mg)

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL deionized water for corrosion test, 25 µL PBS for iritation test
Duration of treatment / exposure:
CORROSION TEST
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test item application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (incubation: 3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid ground test material was applied with a sharp spoon and homogeneously distributed with the vehicle. Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC).

IRRITATION TEST
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively; incubation duration was 1 hour. 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid ground test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
Observation period:
CORROSION TEST
The tissues were washed with PBS to remove residual test item 3 minutes or 1 hour after start of the treatment. The assay medium was then replaced by main reagent 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and tissues were incubated for 3 hours.
After incubation and washing, the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

IRRITATION TEST
The tissues were washed with sterile PBS to remove residual test item 1 hour after start of the treatment. The tissues were then incubated in fresh medium at 37°C for 24 ± 2 hours, were transferred into new 6-well plates with fresh medium and again, were incubated for additional 18 ± 2 hours.
The medium was replaced by 0.3 mL of MTT and the tissues were incubated again for 3 hours.
After incubation and washing, the formazan that was metabolically produced by the tissues was extracted by isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.
Number of animals:
not applicable
Details on study design:
NUMBER OF TISSUES PER TEST
For the corrosion test, 2 tissues were used for 3 min and 1 hour incubation, respectively.
For the irritation test, 3 tissues were used for 1 hour incubation each.

SCORING SYSTEM
- Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water (see table1).
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS (see table2).

EVALUATION OF RESULTS
A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a testitem is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%. Regarding the classification of the test item according to the the severity of the findings, itis suggested to use the most stringent category for test substances leading to viabilities below 50% after 3 min treatment.
A chemical is considered as "irritant", if the mean relative tissue viability with a test item is less than or equal to 50%.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: viability (MTT) %
Basis:
mean
Time point:
other: 3 min
Score:
97
Remarks on result:
other: corrosion test
Irritation parameter:
other: viability (MTT) %
Basis:
mean
Time point:
other: 1 hour
Score:
107
Remarks on result:
other: corrosion test
Irritation parameter:
other: viability (MTT) %
Basis:
mean
Remarks:
tissue 1 - 3
Time point:
other: 1 hour + 42 hours
Score:
99
Remarks on result:
other: irritation test
Irritant / corrosive response data:
CORROSION TEST
The mean viability of the test item treated tissues determined after an exposure period of 3 minutes was 97%, and it was 107% after 1 hour exposure.
IRRITATION TEST
The mean viability of the test item treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 99%.
POSITIVE CONTROLS
The positive controls for corrosion and irritation showed clear effects.
Other effects:
none

Any other information on results incl. tables

Table3: Results for Corrosion Test/Irritation Test

 CORROSION - viability [% of NC]     Exposure: 3 min        Exposure: 1 hour      
Test substance   tissue 1  tissue 2  mean  tissue 1  tissue 2  mean
 NC    101.7  98.3  100  100.4  99.6  100
 test item    95.8  98.1  97  109.5  104 .3  107
 PC     29.7  22.2  26  8.4  8.1  8
                
 IRRITATION - viability [% of NC]    tissue 1  tissue 2  tissue 3  mean    
 NC    97.5  110.7  91.8  100    
 test item    95.0  95.8  107.6  99    
 PC    8.8  9.0  9.1  9    

NC = negaive control; PC = positive control

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
DSD: not classified
CLP: not classified