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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start : 11 March 2013 Completion : 19 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Description:
Clear amber slightly viscous liquid
Batch: 18515
Purity: 100% UVCB

Test animals

Species:
other: adult human-derived epidermal keratinocytes
Details on test animals and environmental conditions:
See details on cell culture in Details on Study Design section

Test system

Vehicle:
unchanged (no vehicle)
Details on study design:
EPISKIN Small Model (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009) was used in the study. 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.

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France. 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 74 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 25 μl 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.

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. Twenty five μl of the undiluted test substance was added (with a pipette) 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 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.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: cell viability
Value:
ca. 102
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: other: no adverse effect therefore reversibility is not relevant. Remarks: The test substance showed 102% cell viability compared to controls and was therefore not irritating. (migrated information)

In vivo

Irritant / corrosive response data:
Alkylated Naphthalene was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Alkylated Naphthalene did not interact with MTT.

The mean absorption at 570 nm measured after treatment with Alkylated Naphthalene and controls are presented below:

Negative control 1.069 ± 0.113
Test Substance 1.094 ± 0.032
Positve control 0.207 ± 0.013

The mean tissue viability obtained after 15 minutes treatment with Alkylated Naphthalene compared to the negative control tissues is shown below:

Negative control 100 %
Test substance 102 %
Positive control 19 %

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Since the mean relative tissue viability for FAlkylated Naphthalene was above 50%, Alkylated Naphthalene is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 19%. The absolute mean OD570 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 was less than 13%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: EPISKIN Small Model
Conclusions:
Alkylated Naphthalene is non-irritant in the in vitro skin irritation test.
Executive summary:

An in vitro skin irritation test was conducted with Alkylated Naphthalene using a human skin model.

This study evaluates the ability of Alkylated Naphthalene to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of Alkylated Naphthalene 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.

Batch 18515 of Alkylated Naphthalene was a clear amber slightly viscous liquid with a purity of UVCB (100%). Alkylated Naphthalene was applied undiluted (25 μl directly on top of the skin tissue for 15 minutes. After a 2 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 minutes treatment with Alkylated Naphthalene compared to the negative control tissues was 102%. Since the mean relative tissue viability for Alkylated Naphthalene was above 50% after 15 minutes treatment Alkylated Naphthalene is considered to be non-irritant.

The positive control had a mean cell viability of 19% after 15 minutes exposure. The absolute mean OD570 (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 was less than 13%, indicating that the test system functioned properly.

It is concluded that this test is valid and that Alkylated Naphthalene is non-irritant in the in vitro skin irritation test under the experimental conditions of the study.