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Description of key information

Two studies were carried out using the EpiDerm Skin Model for the Skin Irritation test and the EpiDerm Corrosion test. Both indicate that the test material is both non-corrosive and non-irritating to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6th October to 18th December 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
The EpiDerTM Kit (MatTek Corporation) was used in this study. The MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”
Justification for test system used:
The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”
Vehicle:
unchanged (no vehicle)
Details on test system:
The MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”.
The method utilizes a 3-minute exposure for a corrosive classification and a 60-minute confirmatory exposure for materials found to be non-corrosive at the 3-minute exposure. Viable
cells reduce the yellow, soluble, oxidized form of the MTT to the blue-black, insoluble, reduced form. The reduced dye is extracted from the tissue with isopropanol, and the amount of reduced
dye is determined spectrophotometrically. The relative viability of the treated tissues is calculated as a percentage of the negative control viability from the absorbance data by dividing
the corrected test article-treated tissue absorbance by the corrected control tissue absorbance, and multiplying by 100. Test materials which reduce tissue viability to <50% within 3 minutes are
considered corrosive by this method. In addition, test materials which result in tissue viability of ≥50% after a 3-minute exposure, but result in tissue viability of <15% after a 60-minute exposure
are also classified corrosive. Test materials which result in tissue viabilities of ≥50% after a 3minute exposure and ≥15% after a 60-minute exposure are classified non-corrosive. Furthermore,
sub-classification of corrosive materials is possible using the 3 minute exposure time as follows:
a sub-category classification of 1A is assigned if the viability is <25%, and 1B/1C if the viability is ≥ 25%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
25mg of test article (100% concentration)
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
The tissues exposed for 3 minutes were held at room temperature.
The tissues exposed for 60 minutes were incubated at standard culture conditions until the completion of the exposure time.
Number of replicates:
2 for 3minutes and 2 for 60 minutes
Details on study design:
The test and control articles were tested by treating four EpiDerm™ tissues per material. Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. Fifty (50) microliters of each liquid control were applied topically on the EpiDerm™ tissue. Twenty-five mg of solid (powdered) test article were similarly applied. Each EpiDerm™ tissue treated with a solid test article also received 25 µL of sterile, deionized water applied directly onto the test article. The test article was gently mixed, and spread over the tissue surface using a sterile bulb-headed rod if needed. The three-minuteexposure time began as soon as the material was spread onto the tissue. This short exposure time precluded treating more than a small number of tissues at once. The tissues exposed for 3 minutes were held at room temperature during dosing, while the tissues exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

A 1.0 mg/mL solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use. Three hundred (300) µL of MTT reagent solution were added to
designated wells in a pre-labeled 24-well plate. The plate was held in the incubator until tissues were added. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium was decanted. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.

After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for 2 - 3 hours at room temperature.

At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed and
200 µL were transferred to the appropriate wells of a 96-well plate. Two hundred (200) µL of isopropanol were placed in the two wells designated as the blanks. The absorbance at 550 nm
(OD550) of each well was measured with a Molecular Devices Vmax plate reader.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
LSN584368 3 minute assay
Value:
ca. 95.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
LSN584368 60 minutes
Value:
ca. 91.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Interpretation of results:
GHS criteria not met
Conclusions:
Non corrosive
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30th October to 18th December 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EpiDerm Skin Kit (MatTek Corporation) was used for the test.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm Skin Kit (MatTek Corporation) was used for the test. After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing.
The EpiDerm™ tissues were treated in triplicate with each test article for 60±1 minutes. Since each test article was a powder, immediately before application of the solid test article, each tissue surface was moistened with 25 µL of sterile CMF-DPBS to improve contact of the tissue surface with the test chemical. After adding the CMF-DPBS, 25 mg of the test article was added to each of three tissues at 1 minute intervals per tissue using a 25 mg sharp spoon (Aesculap #FK623R). The sharp spoon was filled with the test article and then the spoon was leveled. After the three tissues were dosed with the test article, the test article was gently mixed and spread over the tissue surface using a sterile bulb-headed rod. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35±1 minutes, all of the plates were removed from
the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.
After 60±1 minutes of test or control article exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the control articles where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per
treatment group (test article, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of CMF-DPBS and specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMFDPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was visually observed for residual test article using a dissecting scope. In cases where residual test article was observed, sterile cotton-tipped applicators pre-moistened with CMF-DPBS were used to attempt to remove any residual test article from the tissue surface. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the incubator and the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the incubator at standard culture conditions for an additional 18±1 hours for the remainder of the 42±2 hour post-treatment
expression incubation.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
25mg neat test article
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
Incubated for 35 minutes after which removed from incubator and place into a laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.
Number of replicates:
Done in triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of three runs
Value:
ca. 106.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Interpretation of results:
GHS criteria not met
Conclusions:
Non corrosive
Executive summary:

The test article was tested in EpiDerm TM Skin Model for the Skin Irritation Test. Three tissues were used to assess viability after 60minute exposure. Negative and positive controls were tested in parallel. The test articles were not observed to directly reduce MTT in the absence of viable cells. The test material is deemed to be non-irritating to skin (GHS "No category)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification