Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September 2009 to 21 January 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Draft version 6 second circulation followed (20 March 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
The human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, HEP B and HEP C tests are carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells. After a stage of immersed culture of the keratinocytes on the collagen support (3 days), during emersion (10 days) the epidermis differentiates and a horny layer is formed. The production of EpiSkin® is in accordance with the quality reference norms ISO 9001, ensuring traceability and reproducibility of the epidermal tissue as well as that of the quality control tests carried out on each batch of epidermis. The reproducibility of each batch is checked by histological analysis taking into account the general organisation, the stratification of the epidermis, the nucleation of the basal layer and the size of the intercellular spaces, adhesion of the basal layer to the support and the quantity of granular cells and the thickness of the horny layer. Each criterion is scored out of 4. The maximum score is 28. The minimum score for the criterion of acceptability of the model is 19.5. The score for the batch used on this study (09-EKIN-033) is 22.3 mg/mL; standard deviation of 0.3 and CV of 1.2%. The reproducibility of the response of each EpiSkin® batch is tested against a reference irritant: SDS. The IC50 of the surfactant is measured by the MTT assay after 18 h of contact. The minimum score for acceptability of the model is 1.5 mg/mL. The IC50 value for the batch used on this study (09-EKIN-033) is 2.3 mg/mL.

Prior to conducting the irritation assay, it was first necessary to determine if the test item was itself capable of reducing methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite. Since the assay depends on the ability of viable skin cells to reduce MTT to formazan, any reduction by the test item would interfere with the integrity of the results. The test item (10 μL) was added to MTT (2 mL, ca 0.3 mg/mL) in PBS in a glass universal vial (3 replicates) and incubated at ca 37°C in a humidified atmosphere with 5% CO2 for ca 3 h. Formazan production was assessed by visual inspection. Three replicates of the positive control (eugenol, 10 μL) and the negative control (sterile, ultra-pure water, 10 μL) were tested in parallel to demonstrate the efficacy of the MTT solution.

After arrival at Charles River, the EpiSkin® kits were inspected for quality. The pH and temperature indicators were both acceptable. Each individual unit was then transferred to a single well of a 12 well microtiter plate containing maintenance medium (2 mL). The maintenance medium had been warmed to ca 37°C in a waterbath. Prior to dosing, all units were then incubated for ca 24 h at ca 37°C in a humidified atmosphere with 5% CO2.

DMP Tech was applied onto the exposed surface of three viable EpiSkin® reconstructed human epidermis units using a Gilson positive displacement pipette set to deliver 10 μL. The test item was gently spread over the entire exposed skin surface using the tip of the pipette, taking care to avoid damaging the epidermis. The surface area of the EpiSkin® is ca 0.38 cm2 and therefore the application rate was ca 26.3 μL/cm2.

After exposure to the test item or positive (5% sodium dodecyl sulphate) and negative controls (PBS solution) for ca 15 min, the EpiSkin® units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). 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 in assay medium (ca 0.3 mg/mL, 2 mL per well). 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) 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL of neat test material over a surface area of approximately 0.38 cm2, giving an application rate of 26.3 µL/cm2.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
Approximately 42 hours
Number of replicates:
Three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
87.91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
In the MTT direct reduction test, the visual assessment of the formation of the purple-coloured formazan was assessed. 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 DMP Tech did not reduce MTT to formazan after 3 hours incubation.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results of this study do not indicate that Dimethylolpropane Tech (DMP Tech) is a skin irritant.
Executive summary:

An in vitro assessment of irritation potential of skin was conducted using the EpiSkin test system. The method followed was the draft version of OECD guideline 439. Dimethylolpropane Tech (DMP Tech) was exposed for 15 minutes at an application rate of 23.3 µL/cm2. DMP Tech showed a viability of 87.91% when compared to the concurrent negative control.