Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from April 20, 2012 to June 18, 2012

Reliability:

1 (reliable without restriction)

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified

EC Number:

939-273-4

Molecular formula:

CnHxCly(SO3Na)z • n= 14-17 • z < 0,5 • y = 0 ÷ 2,7

IUPAC Name:

Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

- Model used: SkinEthic reconstructed human tissue model EPISKIN
- Source: SkinEthic Laboratories
- Details: 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.
- 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.

Preliminary test
Direct MTT reduction test (Step 1): Non-specific reduction of MTT was evaluated as follows:
two mL of MTT Ready-to-use Solution was incubated with 20 mg of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
Colouring potential test (Step 2): Chemicals' colouring potential was assessed for potential interaction with the test system.
10 mg of the test item was added to 90 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes.
Colouring of the solution at the end of the incubation time was evaluated.

Sample Test System Treatment Amount/well Number of replicates Sample code
Negative
control Live tissue D-PBS 20 µL 3 N1-N3
Positive
control Live tissue 5% SDS in water 20 µL 3 P1-P3
Test item Live tissue PARAFFIN WAXES
AND HYDROCARBON
WAXES, CHLORO,
SULFOCHLORINATED
SAPONIFIED (C14-C17)
SSP-SAMPLE1 20 µL* 3 F1-F3
*:Even if the test item was a dense cream, it was possible to measure the amount in volume (differently from what carried out in the preliminary phase) using a small syringe of adeguate capaci.

calculation
BLANK negative control positive control test item

0.048 0.831 0.085 0.121
0.054 0.82 0.065 0.112
0.053 0.868 0.07 0.06
0.054 0.92 0.064 0.062
0.049 0.829 0.084 0.037
0.047 0.882 0.074 0.066

Mean 0.051 0.858 0.074 0.076
SD 0.003 0.039 0.009 0.033
CV 5.90% 4.60% 12.20% 43.40%

MTT ASSAY:
The tissue insert and controls were incubated with 2 mL well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity.
At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol.
Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction.
At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 µL) of acidic isopropanol were analyzed and used as blank.
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.

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 commercial 10% (w/v) SDS solution in water (SIGMA, batch 088K8703)
Negative control item: D-PBS (GIBCO, batch 1098734).

Control samples:

no

Amount/concentration applied:

20 µL/ epidermis unit

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours recovery period

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 3.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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:

other: Category 2 (irritant) based on CLP criteria

Conclusions:

PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1 has been judged as irritant by in vitro treatment.

Executive summary:

The potential of the test item PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE l 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.