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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: RCX 15-116
Batch No.: 15047AE2
Purity: > 99%
CAS No.: 15305-07-4
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT: solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: the routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
The EpiDerm Skin Model is a well established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.
Vehicle:
water
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm™ (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell™). The EpiDerm™ skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Upon receipt of the EpiDerm - kit, the tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 900 µL fresh assay medium. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared by mixing the MTT concentrate with the MTT diluent and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 6-well plate was incubated at 37 ± 1 °C, 5.0% CO2 / 95% air.
3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.

After 3 min of application, with forceps, the first insert was removed from the 6-well plate. with forceps Using a wash bottle the tissue was gently rinsed about 20 times with PBS (phospate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner. Then the inserts were transferred into a prepared 24-well “MTT assay plate“ containing“containing 300 µL prewarmed MTT solution. The plate was incubated for 3 h at 37 ± 1 °C, 5.0% CO2 / 95% air. 3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into new 24-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer.
Amount/concentration applied:
25 mg of the test item was applied directly atop the EpiDerm™ tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The volume of H2O was increased and the test item was spread to match size of the tissue.
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min and 60 min exposure time).
Number of replicates:
2 replicates for each treatment period

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
>= 50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
>= 15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show corrosive effects in the.EpiDerm™ model, a reconstituted three-dimensional human epidermis model.
Executive summary:

In the present study, the skin corrosivity potential of RCX 15 -116 was analysed. Since corrosive chemicals are cytotoxic to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm™, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was³ 50% (97.2%) after 3 min treatment and³ 15% (104.9%) after 60 min treatment. The controls confirmed the validity of the study. The mean OD570of the two negative control tissues was 0.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (6.5%) after 60 min treatment. In the range of 20 – 100% viability, the coefficient of variation (CV) of replicate tissues of all dose groups was£ 30% (0.2% - 12.9%).

In conclusion, under the given conditions, the test item showed no corrosive effects. The test item is therefore classified as "non-corrosive".