Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from April 20,2012 to June 18,2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material: SSP-SAMPLE 1

Test animals

Species:
other: SkinEthic reconstructed human tissue model EPISKIN
Strain:
other: SkinEthic reconstructed human tissue model EPISKIN
Details on test animals and environmental conditions:
TEST System
- Source: SkinEthic Laboratories

- Storage: According to the supplier procedure, the test system was shipped on Monday and received on Tuesday. At arrival, the plate was opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate (supplied) in which each well had previously been filled with 2 ml/well SkinEthic Maintenance Medium.

- Pre-treatment incubation period: Culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

Test system

Type of coverage:
other: no coverage is necessary
Preparation of test site:
other: - Pre-treatment incubation period: Culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.
Vehicle:
unchanged (no vehicle)
Controls:
no
Duration of treatment / exposure:
test item 15 minutes
Observation period:
A 42 hour recovery period was allowed by incubation at 37°C, 5% C02 and saturated humidity.
Number of animals:
in vitro test
Details on study design:
TEST SITE
The test site consists of an airlifted, living, multi-layered tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionality equivalent to human tissue in vivo.
Normal human keratinocytes are used to reconstruct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker chemicals.


REMOVAL OF TEST SUBSTANCE
- Washing:At the end of the exposure, the test item was mechanically removed from the surface and each tissue was rinsed three times with approximately 25 mL of sterile D-PBS filling and empting the tissue insert.
The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.


- Other: MEDIA
Dulbecco's Phosphate buffered saline (D-PBS)
Sterile water
MTT Reagent
MTT Stock Solution
MTT Ready-to-use Solution
Acidic Isopropanol
(0.04 N HCl in isopropanol)

Positive control item : 5% (w/v) sodium dodecyl sulphate (SDS) solution, obtained by 1:1 dilution in sterile water (Baxter, batch 11K0903) of a sterile commerciai 10% (w/v) SDS solution in water (SIGMA, batch 088K8703)
Negative control item: D-PBS (GIBCO, batch 1098734).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: percentage of relative viability
Value:
ca. 3.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: not applicable. Max. score: 100.0. Reversibility: no data. (migrated information)

In vivo

Irritant / corrosive response data:
After appropriate blank subtractions and/or corrections for the background controls, means, standard deviations, coefficients of variation, mean relative viability values (percentage relative to the negative control) will be calculated.
Cut-off values for the endpoint of the test are established as follows:
Criteria classification
Mean relative viability ≤ 50% Irritant
Mean relative viability > 50% Not irritant

Any other information on results incl. tables

validity criteria:

Negative controls: OD values of the negative control samples 0.600, CV% ≤ 18. Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and CV% ≤ 18. Test item data acceptance: CV% ≤ 18.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified has been judged as irritant by in vitro treatment.
Executive summary:

The potential of the test item Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKINTM

The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

Before the Main Assay, a preliminary test was carried out to assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of colouring water per se.

No interaction was recorded between the test item and MTT in test conditions similar to those of the Main Assay. Moreover, no colouring potential of the test item in contact with water was recorded. Thus, no additional control was added in the main phase for the evaluation of non specific coloration which may influence evaluation of results.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit each measuring 0.38 cm^2 (treatment level: 53 µL/cm^2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco's phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/ epidermis unit.

The negative control gave the expected baseline value and variability, in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (2.9% of cell viability when compared to the negative control) and variability (CV% equal to 12.2).

Therefore, the assay was regarded as valid.

The test item induced cell death in the three replicates with a mean cell viability of 3.8% when compared to the negative control. Intra-replicate variability was higher than expected. This may be due to the nature of the substance (cream) and thus, to the fact that residues might be present on the surface even with a higher number of washings after treatment. However, since the behavior of the three replicates indicated that viability was well below the cut-off value of 50%, the test item results were accepted as valid.

According to the established criteria (cell viability less than 50%), the test item is considered to have irritant effect on the skin under the reported experimental conditions.