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Toxicological information

Eye irritation

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Administrative data

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

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 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

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 / 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.
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.

Results and discussion

In vitro

Resultsopen allclose all
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).

In vivo

Resultsopen allclose all
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.

Applicant's summary and conclusion

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.