Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-02 to 2009-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with following deficiencies: Test kit information is missing. No standard deviation [%], no absorbance [%] tissue 1, 2 + 3 and so no max. score.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: OECD 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original guideline adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.46. (IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST), 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Molecular formula: C14H25NO3
- Molecular weight: 255.36g/mol
- Physical state: liquid
- Storage condition of test material: at room temperature, protected from light and moisture

Test animals

Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 µL of the test item were applied to each of triplicate tissues, spread to match the tissue size.






Duration of treatment / exposure:
15 ± 1 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE:
EpiSkin™ Kit (Lot No.: 09-EKIN-029) was purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts. EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on September 01, 2009. On day of receipt EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.

TEST FOR DIRECT MTT REDUKTION
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 µL were added to 1 ml of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTTsolution colour turned blue/ purple, the test item was presumed to have reduced the MTT. A colour change could not be observed.

TREATMENT:
- Prewarming of EpiSkin™ tissues about 24 hours incubation. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1 °C) maintenance medium.
- Prior to the test based on recommandation of the Sponsor, an amount of the test item was warmed in the water-bath to 50 °C to decreases its high viscosity. After cooling this amount of test item to 37 °C, 15 µL were applied to each of triplicate tissues.
- The negative control (deionised water (Lot no. 06.07.09; Volume 15 µL) and positive control (5% SLS (Sodium lauryl sulphate, Sigma 82024 Taufkirchen) solution in deionised water, prepared freshly prior to the performance of the experiment; Volume 15 µL) were added into the insert atop the concerning EpiSkin™ triplicate tissues each.
- The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1 °C, 5 ± 0.5 % CO2.
- After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1 °C, 5 ± 0.5 % CO2.
- After the further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 70 hours extraction of the colorant cells (cell viability test).
- The amount of extracted colorant was determined photometrically at 570 nm.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552).
The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. The MTT concentrate was prepared freshly.

After a 3 hour incubation period (37 ± 1°C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted about 70 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] X 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.

Results and discussion

In vivo

Results
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 15 min incubation
Score:
15.5
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item the relative absorbance values were decreased to 15.5%. This value is well below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Any other information on results incl. tables

HISTOLOGICAL DATA

Positive Control

Negative Control

Number of Studies

49

Number of Studies

49

Period

July 2007 – June 2009

Period

July 2007 – June 2009

Mean Viability

14.3%

Mean OD

1.107

Standard Deviation

11.3%

Standard Deviation

0.287

Range of Viabilities

3% - 32%

Range of ODs

0.7 – 1.6

Results after treatment with the test item

Dose group

Treat-ment Interval

Absor-bance
570 nm
Tissue 1*

Absor-bance
570 nm Tissue 2*

Absor-bance
570 nm Tissue 3*

Mean Absor-bance
of 3 Tissues

Rel. Absor-bance

[% of Negative Control]**

Negative Control

15 min

0.7613

0.6991

1.0835

0.8480

100.0

Positive Control

15 min

0.2468

0.0485

0.0956

0.1303

15.4

BIO 1796

15 min

0.0823

0.0696

0.2417

0.1312

15.5

* Mean of two replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbancetestitem)/(absorbancenegativecontrol)

- After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD0.6 for the 15 minutes treatment interval thus showing the quality of the tissues.

- Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 15.4% thus ensuring the validity of the test system.

- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is irritant to the skin.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is irritant to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is classified as Category 2, H315.