Phosphorous ester

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: OECD 437

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-18

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICCVAM Recommended BCOP Test Method Protocol

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

esterification products of castor oil and polyphosphoric acids

EC Number:

614-406-6

Cas Number:

68308-61-2

Molecular formula:

C57H107P3O18

IUPAC Name:

esterification products of castor oil and polyphosphoric acids

Details on test material:

- Name of test material (as cited in study report): GARDO TP10451
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 2939R
- Expiration date of the lot/batch: 2010-02-18

Test animals / tissue source

Species:

other: bovine eyes

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS) containing the antibiotics Penicillin (100IU/mL) and Streptomycin (100µg/mL) and transported to the laboratory on ice packs. The eyes were used within 5 hours of slaughter.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Number of animals or in vitro replicates:

3 bovine eyes / test material
3 bovine eyes / positive control
3 bovine eyes / negative control

Details on study design:

Preparation of Corneas: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in BCOP holders.

Selection of Corneas and Opacity Reading: The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea. The average opacity for all corneas was calculated.
Three corneas were numerically allocated to the test material. Three corneas were also numerically allocated to the negative control material and three corneas to the positive control material.

The anterior and posterior chambers of each BCOP holder were filled with fresh complete MEM and plugged. The holders were incubated at 32 ± 1ºC for at least 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Test Material Administration and Opacity Measurements: The MEM was removed from the anterior chamber of the BCOP holders. As the test material was viscous the window-locking ring and glass window were removed from all the holders. 0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the applied material over the entire cornea. The window locking ring and glass window were replaced and each holder was incubated in a horizontal position at 32 ± 1ºC for 10 minutes.
At the end of the exposure period the window-locking ring and glass windows were removed from the appropriate chambers. Using a syringe to create a whirlpool effect each cornea was rinsed with fresh complete MEM containing phenol red. Care was taken not to damage the cornea. Due to the viscosity of the test material this method of rinsing was unsuccessful. A further attempt to remove the test material from the cornea was conducted by placing a layer of medium over the cornea. Copious amounts of complete MEM containing phenol red was gently sprayed through the medium layer at the residual test material on the corneas. However, residual test material still remained on the cornea after rinsing. For consistency the control corneas were treated in the same manner. The window locking ring and glass windows were replaced and the anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 +/- 1ºC for 120 minutes +/- 10 minutes.
After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from both chambers (anterior chamber first) was removed. The posterior chamber was refilled with fresh complete MEM and 1 mL of 4 mg/mL sodium fluorescein was applied to the anterior chamber. The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 +/- 1ºC for 90 minutes +/- 5 minutes.

Permeability Determinations: After incubation all the medium in the posterior chamber of each holder was decanted using a syringe with a needle attached and thoroughly mixed in tubes, pre-labelled according to holder number, so that a representative sample was obtained for the OD492 determination.
360 µl of medium representing each cornea was applied to a designated well on a 96 well plate. Two wells were designated as blanks remained empty. These were used for blank subtraction purposes. The optical density at 492nm (OD492) was measured using the Anthos 2001 micro plate reader.
A sodium fluorescein calibration curve was performed to determine the linear range of the testing facilities microplate reader.

Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

EVALUATION OF RESULTS:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group. The opacitometer was calibrated on the day of the test prior to testing (see appendix 2). Calibration of the opacitometer was considered to be acceptable.

Permeability Measurement: The raw OD492 of each well was automatically blank corrected by the Anthos 2001 microplate reader. The OD492 of each well was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
Final Corrected OD492 = OD492 each treated cornea – mean OD492 negative control corneas.

In Vitro Irritancy Score: The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.
Visual Observation: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
The following classification scheme was established by Sina et al. (1995)6 based upon studies with a wide range of test materials.
0 – 25 = mild irritant
25.1 – 55 = moderate irritant
55.1 and above= severe irritant

Results and discussion

In vivo

Irritant / corrosive response data:

The in vitro irritancy score of the test material was 124.9 but found to be inconclusive.

Any other information on results incl. tables

The results of the study were considered to be inconclusive. The test material was an extremely viscous liquid*). At the end of the exposure period attempts to rinse the viscous test material from the eye were ineffective resulting in residual test material coating the cornea. Therefore, it was considered that the opacity and permeability readings were unreliable. Due to the extremely viscous physical state of the test material, the results of this study were considered inconclusive and, therefore, it was not possible to make a prediction of eye irritation.

*) Remark: Gardo TP10451 is a reaction lubricant. Thus, the high viscosity is a relevant and desired property.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: The test result of the in vitro irritation score of 124.9 were considered inconclusive due to the extremely viscous physical state of the test material. Therefore, it was not possible to make a prediction of eye irritation. As preliminary classification

Conclusions:

As preliminary classification and for precautionary reasons, the substance is classified as irritating to the eyes (R36) until the results of the in vivo study (on-going) are available.

Executive summary:

The study followed the procedures of the ICCVAM recommended protocol for the Bovine Corneal Opacity and Permeability (BCOP) test method. A study was performed to assess the ocular irritancy potential of the test material to the isolated bovine cornea.  The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control materials were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score. 

The in vitro irritancy score of the test material was 124.9 (equivalent to severe eye irritant) but found to be inconclusive. The test material was an extremely viscous liquid. At the end of the exposure period attempts to rinse the viscous test material from the eye were ineffective resulting in residual test material coating the cornea. Therefore, it was considered that the opacity and permeability readings were unreliable. Due to the extremely viscous physical state of the test material, the results of this study were considered inconclusive and, therefore, it was not possible to make a prediction of eye irritation.

As preliminary classification and for precautionary reasons, the substance is classified as irritating to the eyes (R36) until the results of an in vivo study are available.