Registration Dossier

Administrative data

Description of key information

In-vitro Skin irritation, OECD 439: non-irritant
In-vitro Eye irritation, OECD 437: non-irritant
Respiratory irritation: not tested
The in-vitro skin corrosion study (required in section 8.1) Annex VII is not proposed by the registrant as the test carried out according to OECD 473 showed that the test item DICYCLOHEXYLPHTHALATE is not classified as corrosive or severely irritant to the eye as well as the vitro skin irritation study carried out using a reconstructed human epidermis (RhE) showed that the test item is considered not irritant.
Therefore, on the basis of the results obtained in the above mentioned studies, no additional in vitro testing should be needed to identify the corrosivity potential of DICYCLOHEXYLPHTHALATE.
The in-vivo skin irritation study (required in section 8.1.1) Annex VIII is not proposed by the registrant as all the in-vitro studies on skin and eye irritation/corrosion tests are clearly negative and therefore it is expected that the substance doesn't pose any risk.
The in-vivo eye irritation study (required in section 8.2.1) Annex VIII is not proposed by the registrant since all the in-vitro studies on skin and eye irritation/corrosion tests are clearly negative and therefore it is expected that the substance doesn't pose any risk.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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
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.
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.





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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized guidelines
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 Guideline 113 Supplement to Test Guidelines 437 and 438: The Bovine Corneal Opacity and Permeability and Isolated Chicken Eye Test Methods: Collection of Tissues for Histopathological Evaluation and Collection of Data on Non severe Irritants.
Deviations:
no
GLP compliance:
yes
Species:
other: Bovine
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Slaughter house: Butcher Service s.r.l.- Mattatoio no. 2067 M
St. Teverina Km. 7800 - 01100 Viterbo
Age of animals : 6-12 months
Vehicle:
physiological saline
Controls:
other: Physiological saline (0.9% NaCl; BieffeMedital batch no. 10I0208) as a control of the test system.
Amount / concentration applied:
20% (w/v) being the test item a solid non surfactant.
Duration of treatment / exposure:
Corneas were exposed in horizontal position for 4 hours ± 5 minutes, incubated in a liquid bath at 32 ± 1°C.
Observation period (in vivo):
The corneas in their holders were incubated in a liquid bath at 32 ± 1°C at least for 1 hour to permit metabolic stabilisation.
At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without phenol red maintained at approximately 32°C (posterior first).
Complete EMEM*
- without phenol red: EMEM* Gibco (Invitrogen) Cat. no. 51200 (batch no. 998577) supplemented with 1% (v/v) Foetal Bovine Serum (FBS - Hyclone - batch no. ATJ33161)


-with phenol red: EMEM* Gibco (Invitrogen) Cat. no. 21090 (batch no. 910192) 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.

Number of animals or in vitro replicates:
3 corneas for the test item, 3 corneas as negative controls treated with physiological saline 0.9 % NaCl, 3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl .
Details on study design:
Examination: Eyes were examined for the presence of any defects which could render the eye unsuitable for use (opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation).
Cornea excision: Each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed into a Petri dish containing HBSS.
Mounting in the chamber: Each cornea was mounted into a prewarmed testing chamber (see Figure 1) with the endothelial surface of the cornea placed in contact with the O-ring of the posterior part of the chamber. Care was taken to ensure the correct position of the cornea minimizing the presence of pigmented area inside the O-ring and avoiding movements which would damage the cornea.
The chamber was then filled with complete EMEM without phenol red maintained at 32 ± 1°C (posterior part of the chamber first to maintain convexity).
Equilibration: The corneas in their holders were incubated in a liquid bath at 32 ± 1°C at least for 1 hour to permit metabolic stabilisation.
At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without phenol red maintained at approximately 32°C (posterior first).
Selection : Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer).
During measurement, attention was paid to keep corneas away from the bath for the minimal time needed.
Corneas with a basal value ≥ 7 arbitrary units are excluded from testing.
The mean opacity of the remaining corneas was determined. Corneas were distributed in treatment groups starting from those nearest to the mean value for the negative control group and trying to approximately maintain opacity values similar to the mean of the negative control group in the treatment and positive control groups.
Irritation parameter:
cornea opacity score
Value:
0.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer).
Irritation parameter:
other: cornea permeability
Value:
1.67
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer).
Irritation parameter:
other: cornea opacity
Basis:
mean
Score:
0.1
Reversibility:
other: not applicable
Remarks on result:
other: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer).
Irritation parameter:
other: cornea permeability
Basis:
mean
Score:
1.67
Max. score:
80
Reversibility:
other: not applicable
Remarks on result:
other: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer).
Irritant / corrosive response data:
The negative control treatments 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 did not increase the corneal opacity, being the calculated mean value equal to 0. This was confirmed at the macroscopic observation in which the three corneas did not show any visible alteration.
With reference to the cornea permeability, this was not affected when compared to that of negative control, thus indicating no alteration of corneal barrier.

The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 72.7. Opacity was 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 1.6740.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The potential of the test item, DICYCLOHEXYLPHTHALATE, to be severely irritant or corrosive to the eye, was measured by its ability to induce opacity and increase permeability in isolated bovine corneas.
No significant alterations of cornea opacity or permeability were recorded during the study when compared to those of negative control.
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.
Moreover, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is no indication of irritant effect to the eye.
Executive summary:

The potential of the test item, DICYCLOHEXYLPHTHALATE, 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) and OECD Supplement to Test Guidelines nos. 437 and 438.

The test item was tested suspended at 20% (w/v) in physiological saline (being a solid non surfactant) on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.

Positive and negative controls [a 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar condition.

The mean opacity detected with an opacitometer at the end of the test item exposure period was 0.1, comparable to the negative control.

After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability.Mean OD490value of the corneas treated with the test item was unaffected by treatment, as well as the negative control.Negative and positive controls gave the expected results.  

The results obtained indicate that the test item did not induce changes in both cornea opacity and permeability under the reported experimental conditions. The calculatedin vitroirritancy score (IVIS) for the test item is 0.

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

Moreover, according to the criteria stated in theOECD Supplement to Test Guidelines nos. 437 and 438, there is no indication of irritant effect to the eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

An in-vitro test of the registered substance was performed on skin irritation according to OECD Guidelines 439. The study can be ranked as klimisch code 1: reliability without restrictions. The result of the study clearly showed that no toxic effect was found, therefore the irritative effect of dicyclohexylphthalate can be concluded as no irritating.

The in-vitro skin corrosion study is not proposed by the registrant as the test carried out according to OECD 437 showed that the test item DICYCLOHEXYLPHTHALATE is not classified as corrosive or severely irritant to the eye as well as the vitro skin irritation study carried according to OECD 439 showed that the test item is considered not irritant. Therefore, on the basis of the results obtained in the above mentioned studies, no additional in vitro testing should be needed to identify the corrosivity potential of DICYCLOHEXYLPHTHALATE.

Eye irritation:

An in vitro test of the registered substance was performed on eye irritation complying with OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method for identifying ocular corrosives and severe irritants. This study is regarded as key study as it can be ranked as klimisch code 1: reliability without restrictions. The test result showed that dicyclohexylphthalate is not irritating.


Justification for selection of skin irritation / corrosion endpoint:
GLP study in compliance with international recognized guidelines.

Justification for selection of eye irritation endpoint:
GLP study in compliance with international recognized guidelines.

Justification for classification or non-classification

The available studies for eye and skin irritation indicate that the notifiable substance is not irritating to the eyes or skin. Thus, the data are conclusive but not sufficient for classification for eye and skin irritation.

 

No information is available for respiratory irritation, thus the notifiable is not classified as a respiratory irritant as data are lacking.