Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated, saponified

Endpoint summary

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:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

other: SkinEthic reconstructed human tissue model EPISKIN

Strain:

other: SkinEthic reconstructed human tissue model EPISKIN

Details on test animals or test system 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 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation

Remarks:

other: in vitro MTT test

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 3,2012 to March 3,2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliance with international guideline

Qualifier:

according to guideline

Guideline:

other: MTT test method

Deviations:

no

Principles of method if other than guideline:

The method allows to evaluate a toxic event and related cytotoxicity by a colorimetric assay. Solution of MTT in balanced saline is yellowish in color. Mitochondrial dehydrogenase of viable cells cleaves the tetrazolium ring yielding blue/purple MTT crystals which are insoluble in aqueous solutions. Crystals formed by viable cells are retained in the polycarbonate filter used as substrate for the 3D construct. Intense purple colour of the cultures indicates the viability of the cell at the basal layer, whereas colour remains white when necrosis occurs. Negative controls are of a dark blue colour and positive controls are white-yellow.
MTT crystals are extracted by isopropanol and optical density is measured at 570 nm.

GLP compliance:

yes (incl. QA statement)

Species:

other: HCE conrneal epithelium model

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source:SKINETHIC LABORATORIES

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL

- Concentration (if solution):The test item was tested at the dose defined for liquid (30 µL).

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

not required

Number of animals or in vitro replicates:

no aminals required

Details on study design:

The test was performed on triplicate tissues for MTT and in simplicate for histological analysis.
The positive control was Ethanol (99,96%) and as negative control saline solution (NaCl 0,9%) was used. 30 µL of controls (liquids) were directly and uniformly applied topically.

SCORING SYSTEM:cell viability

OTHER: the test was performed in triplicate

Irritation parameter:

other: Cell viability

Basis:

mean

Time point:

other: 1h + 16h

Score:

ca. 0.472

Max. score:

100

Reversibility:

not specified

Remarks on result:

other: SD ± 0.039

Irritant / corrosive response data:

The prediction model for eye irritation ciassification is based on cell viability (MTT-modified protocol) after 1h+16h exposure (SOP M 04).
A cut-off of 50% is accepted for discriminating between potential irritant (<50 %) and not classified (>50 %). Positive control (Ethanol) satisfies acceptance criteria if it is classified as irritant (<50 %).
Mean tissue viability is <50 % Irritant (I) R36
Mean tissue viability is >50 % Not Classified (NC)

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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%).

Executive summary:

The present study has been conducted in order to assess in vitro, on a Human Corneal Epithelium model (HCE), the eye irritation potential of Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified.

Eye irritation potential of the test item was assessed at 1h followed by product washing and a post incubation period of 16h by using the MTT test method to quantify the residual cell viability. A complementary histo-morphological analysis was associated.

According to the adopted prediction model based on MTT results the chemicals have been classified as reported in the following table.

Classification based on cell viability-MTT results.

CHEMICAL	1h+16h
CN	NC
ETOH	IR
S-0512	IR

(NC=Not Classified; IR=Irritant for the eye, R 36 )

The scoring of the complementary histo-morphological analysis was in agreement with the cell viability results.

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%).

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

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.

It can be assumed that the results will be the same also for Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated, saponified. Molecular weight will be usually higher or sulphonation degree, then hydrophylicity and/or salification proportions higher, leading to a better toxicological profile

Justification for selection of skin irritation / corrosion endpoint:
Good in vitro study, GLP with international guideline on a very similar substance

Justification for selection of eye irritation endpoint:
Two GLP in vitro studies have been performed on a very similar substance for skin irritation, a HCE study, that discriminates 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 parameters comes directly from the viability evaluations, leading to classification both for eye and for skin as irritant, with H 315 and H 319