Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Identification: D-C16
Appearance: White crystal
Batch: 14-002
Purity/Composition: 99.3%
Test substance storage: At room temperature, dark place, airtight
Stable under storage conditions until 24 October 2019 (expiry date)

Test animals

Species:
other: skin derived from human adults
Strain:
not specified
Details on test animals and environmental conditions:
Test system: EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 15-EKIN-010).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results
in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:
other: not applicable as in vitro study
Preparation of test site:
other: not applicable as in vitro study
Controls:
other: not applicable as in vitro study
Number of animals:
not applicable as in vitro study
Details on study design:
Preparation and preincubation
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 70 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.2 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Study design
A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.
Test for colour interference by the test substance
D-C16 was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, approximately 10 mg of D-C16 was added to 90 µl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µl Milli-Q water was tested concurrently. At the end of the shaking period colour check is performed. In case the solution turned blue / purple the test substance may have colour interference. A functional test with living skin tissues was performed to show that the test substance did not bind to the tissue and resulted in a false MTT reduction signal.
Test for reduction of MTT by the test substance
D-C16 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 10 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37 °C. A negative control, sterile Milli-Q water was tested concurrently.
In case the test substance reacts with the MTT medium in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues must be used for the cytotoxicity evaluation with MTT.
Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.
Cell viability measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues.Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: optical density 570 nm
Value:
106
Remarks on result:
other:
Remarks:
Basis: mean of 3 experiments. Time point: 15 minutes treatment. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Negative control is set to 100% whereas mean scores for test substance were at 106%, The positive control showed 44%. (migrated information)

In vivo

Irritant / corrosive response data:
D-C16 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that D-C16 did not interact with the MTT endpoint.

Negative control (Phosphate buffered saline (PBS)): Mean tissue viability 100%
Test substance D-C16: Mean tissue viability 106%
Positive control (5% (aq) sodium dodecyl sulphate (SDS)): Mean tissue viability 44%

The results above shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues was 106%. Since the mean relative tissue viability for D-C16 was above 50% D-C16 is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The absolute mean OD 570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with positive control was 27%, which was due to one outlier. However, since the other 2 viabilities were clearly positive and within the historical data range and furthermore the test substance showed a clearly negative result this did not affect study integrity. The standard deviation value of the percentage viability of three tissues treated identically with negative control or test substance was less than 8%.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
D-C16 is non-irritant in the in vitro skin irritation test.
Executive summary:

The study describes the ability of D-C16 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SM TM )). The possible skin irritation potential of D-C16 was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Skin tissue was moistened with 5 µL of Milli-Q water and an excessive amount of D-C16 was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues was 106%. Since the mean relative tissue viability for D-C16 was above 50% after 15 ± 0.5 minutes treatment D-C16 is considered to be non-irritant.

The positive control had a mean cell viability of 44% after 15 ± 0.5 minutes exposure. The absolute mean OD 570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with positive control was 27%, which was due to one outlier. However, since the other 2 viabilities were clearly positive and within the historical data range and furthermore the test substance showed a clearly negative result this did not affect study integrity. The standard deviation value of the percentage viability of three tissues treated identically with negative control or test substance was less than 8%.

Finally, it is concluded that this test is valid and that D-C16 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.