Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Cross-reference
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): XU-18838.00
- Physical state: powder
- Lot/batch No.: 20120135-18
- Storage condition of test material: room temperature, protected from light

Test animals

Details on test animals and environmental conditions:
MatTek Corp. EpiDerm™ reconstituted human epidermis
The EpiDerm™ tissues were stored at 2-8ºC until used. On the day of receipt, an appropriate volume of fresh EpiDerm™ EPI-100-NMM Maintenance Medium was pre-warmed to room temperature. Nine-tenths (0.9) mL of Maintenance Medium were aliquotted into the appropriate wells of each 6-well plate, which were pre-labeled to indicate the test or control substance. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and Millicell® insert prior to opening the sealed package. There was no culture with air bubbles greater than 50% of the Millicell® area used in the assay. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. An appropriate number of tissues were removed from the 24-well shipping plate and transferred to the 6-well plate containing Maintenance Media. The plates were placed into the incubator at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for 60±5 minutes. At the end of the incubation period, the inserts were transferred into wells containing fresh Maintenance Medium and incubated overnight (18 ±3 hours) to acclimate the tissues.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
Duration of treatment / exposure:
60 ± 1 minutes
Observation period:
24 ± 2 hours
Number of animals:
not applicable
Details on study design:
After the overnight pre-incubation, the EpiDerm™ tissues were removed from the incubator and placed into a laminar flow hood at room temperature for at least 5 minutes before treatment initiation. The EpiDerm™ tissues were treated in triplicate with the test substance, XU-18838.00, for 60 ±1 minutes. Since the test substance was a powder, immediately before application of the solid test substance, 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 substance 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 substance and then the spoon was leveled. After the three tissues were dosed with the test substance, the test substance was gently mixed and spread over the tissue surface using a 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 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 exposure, the EpiDerm™ 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 substances 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 substance, 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 CMF-DPBS was decanted. The bottom of the tissue insert was blotted on sterile paper towels and the tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture. The tissue surface was visually observed for residual test substance or excess moisture using a dissecting scope. For the test substance, cotton-tipped applicators pre-wetted with sterile, CMF-DPBS were used to attempt to remove the residual test substance. Once the rinsing was complete, the tissue inserts were transferred to new 6-well plates containing 0.9 mL of fresh Maintenance Medium. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 24±1 hours.
After the 24±1 hour post-treatment expression incubation, the 6-well plates were removed from the incubator. 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 for an additional 18±1 hours post-treatment incubation at standard culture conditions.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
ca. 97.2
Remarks on result:
other:
Remarks:
Basis: other: EpiDerm tissue. Time point: After a 60-minute exposure and a 42-hour post-exposure incubation. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
Based upon the results of this assay, the test substance, XU-18838.00, was predicted to be non-skin irritating.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Based upon the results of this assay, the test substance XU-18838.00, was predicted to be non-skin irritating, and thus would not require classification.
Executive summary:

The MatTek Corporation’s EpiDerm™ reconstituted human epidermis model was used to assess the potential skin irritation of the test substance, XU-18838.00, Lot Number 20120135-18. The skin irritation potential of the test substance was evaluated by measuring the relative cell viability in treated tissues at 42-hour post-exposure incubation after a 60-minute exposure to the test substance, according to the Protocol for: In vitro EpiDerm™skin irritation test (EPI-200-SIT) 1 evaluated and validated by the European Centre for the Validation of Alternative Methods (ECVAM). The protocol met the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)2. Under the conditions specified in this study, the test substance, XU-18838.00, resulted in 97.2% cell viability and was not predicted to be a skin irritant. Results of the positive control and negative control met the criteria of a valid assay. Accordingly, the test substance, XU-18838.00, would be labeled non-irritant to skin (i.e., “no category”) within the Globally Harmonized System, or “No label” within the context of the European Risk Phrase system.