Dicyclohexyl phthalate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexyl phthalate

EC Number:

201-545-9

EC Name:

Dicyclohexyl phthalate

Cas Number:

84-61-7

Molecular formula:

C20H26O4

IUPAC Name:

1,2-dicyclohexyl benzene-1,2-dicarboxylate

Test material form:

other: crystalline powder

Test animals / tissue source

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

Test system

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.

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:

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.

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.

Results and discussion

In vitro

Results

Results 1

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).

Results 2

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).

Applicant's summary and conclusion

Interpretation of results:

other: not irritating

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 calculated in vitro irritancy 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.