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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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)
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.

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).
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)
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

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.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
FromApril 20,2012 to June 14,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complint with international guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment no. 113 - Supplement to Test Guidelines 437 and 438: Collection of Tissues for Histopathological Evaluation and Collection of Data on Non severe Irritants.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eye
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: Butcher Service s.r.l. - Mattatoio no. 2067 M
- Age of animals: 6-12 months
- Killing time: From 9:30 to 11:30 in the morning.
- Transport condition: Maintained in eyes transport solution at approximately 4°C.
- Storage condition: These will be placed into plastic containers with approximately 1 litre of Hanks balanced salt solution (HBSS) for every 25 eyes collected. The time of killing of the animals will be estimated. The containers will be transported to the test facility in refrigerated condition (approximately 4°C) and the time of arrival will be recorded.
- Eye health: all eyes will be carefully examined. Any defects such as opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation will render the eye unsuitable for use
Vehicle:
other: EMEM
Controls:
other: not required
Amount / concentration applied:
Fresh solutions/suspensions of the test item will be prepared for each day's work; solutions/suspensions will be prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item.
- Liquid test items: with the exception of surfactants, will be used as supplied.
- Solid substances: with the exception of surfactants, will be dissolved/suspended to 20% (w/v) concentration in Eagle’s Minimum Essential Medium (EMEM), sterile deionized water or physiological saline (NaCl 0.9%). Paraffin oil may be used for hydrophobic substances. If necessary, they will be ground to reduce particle size and aid suspension.
- Surfactants will be dissolved/diluted to 10% (v/v or w/v) concentration using EMEM, sterile deionized water or physiological saline.
- Surfactant preparations will be tested neat, unless differently indicated by the Sponsor. Dilution/suspensions using EMEM, sterile deionized water or physiological saline may be evaluated



Duration of treatment / exposure:
Corneas were exposed in horizontal position for 10 ± 1 minutes, incubated in a liquid bath at 32 ± 1°C
Observation period (in vivo):
Corneas were maintained in horizontal position, for further 2 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.
Number of animals or in vitro replicates:
Once underwent the first examination, a total of 10 corneas were processed (out of 12 delivered) for a final selection of the 9 required corneas according to basal opacity.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Time after start of exposure:10 minutes
SCORING SYSTEM:according to OECD 437
- Washing: After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red (3 washing with 3 mL each). A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with prewarmed complete EMEM without phenol red.

TOOL USED TO ASSESS SCORE: fluorescein

other:
CONTROL ITEMS
Positive control item: 1% (w/v) sodium hydroxide (NaOH; Sigma, batch no. BCBG2025V) in water (Baxter, batch no. 11K0903), being the test item a liquid.
Negative control item: Physiological saline (0.9% NaCl; BieffeMedital batch no. 10I0208) as a control of the test system.

Media
Eyes transport solution: Hanks balanced salt solution (HBSS) Modified supplemented with Penicillin sulphate and Streptomycin sulphate both at the final concentration of 100 IU/mL.
Complete EMEM*
- without phenol red: EMEM* Gibco (Invitrogen) Cat. no. 51200 supplemented with 1% (v/v) Foetal Bovine Serum (FBS - Hyclone - batch no. ATJ33161)
- with phenol red: EMEM* Gibco (Invitrogen) Cat. no. 21090 supplemented with 1% (v/v) Foetal Bovine Serum (FBS - Hyclone - batch no. ATJ33161)
* also named: Minimum Essential Medium Eagle's (MEM) Modified or MEM or Eagle’s MEM. These media were prewarmed in a water bath at 32°C during the experimental procedure.
Sodium fluorescein solution: Sigma Cat. no. F-6377. This was formulated to give 4 mg/mL solutions in DPBS [Gibco Cat. No. 14190].
Irritation parameter:
other: IVIS
Basis:
mean
Time point:
other: 10 minuts
Score:
ca. 6.1
Max. score:
100
Reversibility:
other: not applicable
Irritant / corrosive response data:
Details of measurements carried out at the observations are reported in Table 2.
The negative control samples did not induce corrosion as the mean opacity and permeability values were homogeneous and in line for the expected values for this kind of control.
The test item slightly increased the corneal opacity, being the calculated mean value 2.1.
The alteration was not homogeneous in the three replicates since in replicate A1 the response was higher than in the remaining two.
At the macroscopic observation the three corneas showed slight opacity.
With reference to the cornea permeability, this was slightly affected when compared to that of negative control, thus indicating a minimal alteration of corneal barrier. Also in this case alteration was higher in replicate A1.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 232.6. Opacity and exfoliation were noted in the three replicates at the end of the 4-hour post-incubation period.
The corneal permeability was also increased. The calculated mean permeability OD490 value was 2.4223.

Analysis of data

For each test point, the mean value of opacity obtained after exposure was calculated. As appropriate, negative control was subtracted from the opacity of the test item or positive control.

The mean value of permeability, expressed as optical density units, was also calculated and corrected against the mean negative control value.

The in vitro irritancy score (IVIS) of the test item was calculated from these values in the following manner:

IVIS = mean opacity score + (15 x mean Permeability OD490 score)

Classification of the test item was performed according to the in vitro irritancy score:

Value Classification

> 55.1 Corrosive or severe irritant

In addition, the test item was evaluated also for the eye irritancy potential according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438:

Value Classification

0 -3 Non eye irritant

3.1 -25 Mild eye irritant

25.1 - 55 Moderate eye irritant

> 55.1 Corrosive or severe irritant

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential of the test item, PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1, to be severely irritant or corrosive to the eye, was measured by its ability to induce opacity and increase permeability in isolated bovine corneas.
The IVIS was 6.1. The negative and positive controls gave the expected results. The test is therefore considered as valid. According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye. However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is indication of mild irritant effect to the eye.
Executive summary:

The potential of the test item, PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 7 September 2009) and OECD Supplement to Test Guidelines nos. 437 and 438.

The test item was used as supplied (being a cream) on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.

Positive and negative controls [a 1% (w/v) sodium hydroxide solution in water and physiological saline alone, respectively] were concurrently tested in the same number of replicates.

The mean opacity detected with an opacitometer at the end of the test item exposure period was 2.1, slightly higher than that of the negative control. The alteration was not homogeneous in the three replicates since in one replicate the response was higher than in the remaining two.

After the determination of opacity, the epithelial surface was treated with a 0.4% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability.

Mean OD490 value of the corneas treated with the test item was slightly affected when compared to that of negative control, thus indicating a minimal alteration of corneal barrier. Also in this case alteration was higher in the same individual replicate.

Negative and positive controls gave the expected results.

The results obtained indicate that the test item induces slight changes in cornea opacity but especially in corneal permeability under the reported experimental conditions.

The calculated in vitro irritancy score (IVIS) for the test item is 6.1.

According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye.

However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is indication of mild irritant effect to the eye.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three in vitro tests have been performed to assess eye and skin irritation.

Considering EPISKIN test, 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.

Similarly, regarding HCE test for eye, a cell viability < 50% corresponding to the cut-off value for eye irritant classification has been quantified for positive control (0,6246%) and test item (0,4719%). Nevertheless a BCOP test has been performed in addition and the calculated in vitro irritancy score (IVIS) for the test item is 6.1.

According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye.

However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is indication of mild irritant effect to the eye.

As a consequence, since it is practically impossible that an eye irritant not corrosive for eye will be corrosive for skin, the skin corrosion test has not been performed and the resulting assessment and classification is reported below


Justification for selection of skin irritation / corrosion endpoint:
Good in vitro study, GLP with international guideline

Justification for selection of eye irritation endpoint:
Two GLP in vitro studies have been performed for eye irritation, a HCE study, that discrimitates between irritant and non irritant substances and a BCOP, to assess the possibile corrosion behaviour of the substance. From the BCOP study the irritation property coming from HCE has been demonstrated, but the corrosion has been definitively excluded

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Only in vitro tests have been performed, all in agreement with OECD standards and in fully replacement of the in vivo tests, therefore no direct correspondence for assessing the irritation potential for eye and skin can be taken from Regulation CE 1272/2008 and their amendments until July 2012 (II and III), but clear paramenters comes directly from the viability evaluations, leading to classification both for eye and for skin as irritant, with H 315 and H 319