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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 April 2012 to 18 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized 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.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystalline powder
Details on test material:
Name : DICYCLOHEXYLPHTHALATE
Alternative names : DCHP
Dicyclo hexyl phthalate solid
Diciclo esil ftalato solido
Ftalato di cicloesile solido
Batch number : 031-12
Expiry date : 27 January 2013
Description (by the Sponsor) : White crystalline powder
Appearance at first use : White crystalline powder
Storage at RTC : Ambient condition
RTC reference number : 13219
Certificate of analysis : Supplied by the Sponsor and presented in Addendum I Test item characterisation : Not undertaken at the testing facility. The determination of the identity, strength, purity, composition, stability was the responsibility of the Sponsor.

Test animals

Species:
other: not applicable
Strain:
other: not applicable
Details on test animals and environmental conditions:
Not applicable the test system EPISKIN is a reconstructed human epidermis (RhE) model.
The test system EPISKIN TM is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis .
The principle of the RhE test method is based on the premise that chemicals are able to penetrate the stratum corneum and irritant chemicals are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS RN 298-93-1] into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Not applicable
Amount / concentration applied:
Prliminary test:
10 mg of test item was used.

Main test:
10 mg of the test item was used.
Duration of treatment / exposure:
15 min
Observation period:
A 42 hour recovery period was allowed by incubation at 37°C, 5% CO 2 and saturated humidity.
Number of animals:
Not applicable
Details on study design:
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 10 mg of test item at 37°C, 5% CO 2 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) : Chemical's colouring potential was assessed for potential interaction with the test system. 10 mg of the test item was added to 90 L 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.

Main Test:
A Main Assay was carried out including the test item, positive and negative controls. The treatment scheme was the following:

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 DICYCLOHEXYL PHTHALATE 20 mg* 3 B1-B3 *: 10 µL/well of sterile water were added

Exposure period : An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature. Adding was carried out staggering samples of approximately 1 minute. Positive control : an intermediate re-spreading was carried out approximately after 7 minutes of incubation.

Washing : At the end of the exposure, each tissue was rinsed twice 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.

Media collection for further analysis : At the end of the incubation period, the plates were shaked on a plate shaker for about 15 minutes at approximately 300 rpm/min. A volume of 1.6 mL of each incubation medium was removed and stored at -20°C for possible future analysis. No analysis was carried out on these samples, in agreement with the Sponsor.

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% CO 2 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 L) of acidic isopropanol were analysed and used as blank.





Results and discussion

In vivo

Results
Irritation parameter:
other: cell viability
Basis:
mean
Remarks:
compared to the negative control
Time point:
other: not applicable
Score:
92.2
Max. score:
100
Reversibility:
other: not applicable
Irritant / corrosive response data:
The test item did not induce cell death, with a mean cell viability of 92.2% when compared to the negative control. The intra-replicate variability was slightly higher than expected (20.1% instead of 18%). However, since the test item did not clearly induce cell death and the relative cell viability was much higher than the cut-off value (50%) in each of the three replicates, the result was accepted as valid.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The potential of the test item DICYCLOHEXYLPHTHALATE to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN TM . According to the histotoxic potential, when referred to the negative control, the test item is considered to have no effect on the test system (92.2% of mean cell viability when compared to negative control). The negative and positive controls gave the expected results and the study was accepted as valid. According to the criteria defined in the guideline for this test (cell viability less than 50%), the test item is not considered to have irritant effect on the skin under the reported experimental conditions.
Executive summary:

The potential of the test item DICYCLOHEXYLPHTHALATE (batch: 031-12) to be irritant to the skin was investigated through anin vitroskin 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 waterper 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 mg/epidermisunit each measuring 0.38 cm2(treatment level: 53 mg/cm2). 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 L/epidermisunit.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 did not induce cell death, with a mean cell viability of 92.2% when compared to the negative control. The intra-replicate variability was slightly higher than expected (20.1% instead of < 18%). However, since the test item did not clearly induce cell death and the relative cell viability was much higher than the cut-off value (50%) in each of the three replicates, the result was accepted as valid.

According to the established criteria (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.