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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:
28 September to 5 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP study conducted according to current guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Draft OECD Guideline. In Vitro Skin Irritation: Reconstructed Human Epidermis (RHe) Test Method. 20 March 2009 (Version 6) 2nd Circulation.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Di-Penta 93, batch no. 3810066489, a white crystalline powder, was received from Perstorp Holding AB, and was stored protected from light at ambient room temperature. The Sponsor supplied Test Item Data Sheet stated that the batch was produced on 15 December 2008 and “expects not to expire within at least five years after production. The shelf-life is two years from dispatch.”

Test animals

Species:
human
Strain:
other: not applicable.
Details on test animals and environmental conditions:
This was an in vitro study using the EpiSkin system, produced from human keratinocytes obtained from healthy donors..

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable, the study was in vitro
Vehicle:
water
Controls:
other: The negative control was phosphate-buffered saline, the positive control was 5% sodium dodecyl sulphate (SDS)
Amount / concentration applied:
10 mg ± 2mg was applied onto the exposed surface of the EpiSkin. The surface of the EpiSkin was moistened with sterile, ultra-pure water (5 µL) prior to dosing. The surface area of the EpiSkin was ca 0.38 cm² and therefore the mean application rate was ca 23.6 mg/cm².
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Three viable EpiSkin units
Details on study design:
After exposure to the test item or positive and negative controls for ca 15 min, the EpiSkin units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). After washing the units were blotted dry with tissue paper and transferred to fresh maintenance medium. After washing, all skin units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 42 h at ca 37°C in a humidified atmosphere with 5% CO2.

After the recovery period, all EpiSkin units were transferred to a new 12 well microtiter plate containing MTT solution (2 mL per well, ca 0.3 mg/mL) in assay medium. Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The skin units were then incubated for ca 3 h at ca 37°C in a humidified atmosphere with 5% CO2. After incubation, each skin unit was removed from the MTT solution and gently tapped dry on tissue paper to remove any excess moisture. The exposed skin was then completely removed from the unit using a specially designed biopsy punch. The epidermis was separated from the collagen matrix and both layers added to an appropriately labelled microfuge tube containing acidic isopropanol (500 μL). Samples were then stored at ca 4°C protected from light.
After ca 72 h storage, the samples were removed from the refrigerator and mixed by vortexing to ensure a homogenous mixture. Two aliquots (200 μL) of each sample were then added to a 96 well flat bottom microtiter plate for each sample, ensuring that no solid material was removed from the sample tube. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background acidified isopropanol sample contained on the plate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
90.29
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hours. Max. score: 94.39. Reversibility: other: not applicable. Remarks: SD 5.19%. (migrated information)

In vivo

Irritant / corrosive response data:
The negative control results were within the acceptance criteria defined in the ECVAM validation SOP. The positive control results were within the acceptance criteria defined in the ECVAM validation SOP. Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative
control value. Percentage viabilities are shown in Table 1.
Other effects:
No other effects reported.

Any other information on results incl. tables

MTT Direct Reduction Test:

The test was scored by visual assessment of the formation of the purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to formazan almost immediately, generating a dark purple colour before incubation. The negative control (sterile, ultra-pure water) and Di-Penta 93 did not reduce MTT to formazan after ca. 3 h incubation.

Viability of EpiSkin Cultures

Test item

Mean Viability (%) after 42 hours

SD (%)

CV (%)

Di-Penta 93

90.29

5.19

5.75

5% SDS Solution (positive control)

10.40

4.10

39.42

PBS Solution (negative control)

100.00

7.82

7.82

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information GHS no category Criteria used for interpretation of results: EU
Conclusions:
Di-Penta 93 is non-irritant when tested within the EpiSkin® in vitro irritation assay.
Executive summary:

Di-Penta 93 was tested for dermal irritation in vitro, using the SkinEthic EpiSkin® in vitro irritation assay. The endpoint of the assay was the estimation of cell viability by assaying the reduction of methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite by mitochondrial reductase. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value. A preliminary test was conducted to assess the ability of Di-Penta 93 to directly reduce MTT to formazan. Di-Penta 93 did not interact with the MTT solution. The irritation potential was assessed by applying Di-Penta 93 (ca 10 mg) directly onto the surface of three viable EpiSkin® reconstructed human epidermis units for ca 15 min. Di-Penta 93 was then washed from the surface of the EpiSkin® and the units returned to the incubator for a recovery period of ca 42 h. After the recovery period, the skin units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for ca 3 h. Biopsies of the EpiSkin® membranes were then removed and added to acidic isopropanol. The formazan production was assessed by measuring the optical density of the extract at 550 nm and the viability of each individual tissue was calculated as a percentage of the mean negative control viability. Exposure to Di-Penta 93 resulted in an EpiSkin® viability of 90.29% ± 5.19% of the negative control value. The positive and negative controls, conducted in parallel, were within the defined acceptance criteria and demonstrated the efficacy of the test system.

In conclusion, Di-Penta 93 is non-irritant (“no category” in accordance with GHS classification) when tested within the EpiSkin® in vitro irritation assay.