Copper, .beta.-resorcylate salicylate lead complexes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2010 to 21 october 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD n° 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

not applicable

Principles of method if other than guideline:

The method used is adapted from that described by Gautheron P. & al. (1992) Fundam. Appl. Toxicol. 18 442-449.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper Lead Resorcylate Salicylate Complex

EC Number:

614-455-3

Cas Number:

68411-07-4

Molecular formula:

C7H5O4-, C7H5O3- Cu , Pb

IUPAC Name:

Copper Lead Resorcylate Salicylate Complex

Details on test material:

- Name of test material (as cited in study report): Lead-Cooper-Resorcylate-Salicylate (LC 12-15)
- Physical state: light pale green powder
- Composition of test material, percentage of components: 11.9% of copper, 35.7% of lead, 14.1% of resorcylate and 35.7% of salicylate
- Lot/batch No.: 09/201 (on the labeling), PRESE123/ (on the analytical certificate)
- Expiration date of the lot/batch: 04 November 2011
- Storage conditions of test material: at room temperature.

Test animals / tissue source

Species:

other: isolated calf cornea

Strain:

other: bovine calf

Details on test animals or tissues and environmental conditions:

- Source: Origin: calf eyes were obtained from freshly slaughtered calves at the abattoir SOCAVIA, Cany Barville, France.

- Reason for choice: calf corneas are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

- Transport from supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing antibiotics (Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin).

- Preparation of the corneas: the corneas were prepared as quickly as possible after receipt. Each step was carried out avoiding to touch the corneas in order to not injure them

- Selection: upon arrival at CIT, all eyes were carefully examined macroscopically for defects (opacity, scratches, pigmentation, etc) and those exhibiting any defect were discarded. The too large eyes were also discarded in order to avoid the formation of folds at the assembly of corneas in the holder. The examination was performed under a lamp and using HBSS in order to maintain the corneas moistened and shiny. Each cornea was observed with attention, while making swivel the eye in order to see any less refringent areas under the light or any scratches.

- Preparation of the selected corneas: the tissue surrounding the eyeball was carefully pulled away and the cornea was dissected such that approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were stored in HBSS until all corneas were dissected.
As the corneas were not used extemporaneously for a treatment, they were washed three times of 15 minutes each in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature, then stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C for 24 hours maximum before use.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): as the dosage form was not able to be sampled by a pipette, an amount of 750 mg ± 75 mg of the test item was gently applied to the cornea, as uniformly as possible.

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Three corneas were used for each treated series (test item, positive control and negative control).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): as the dosage form was solid, the dosage form having adhered to the walls of the compartment was eliminated using a cotton bud. Then the compartment was filled with heated cMEM (32°C). The rinsing was repeated five times (i.e. until the dosage form is completely removed from the compartment). The efficiency of the rinsing was visually appreciated with the transparency of the medium of rinsing (despite the rinsing performed, residual test item was noted over corneas after treatment).

TOOL USED TO ASSESS SCORE: opacitometer, fluorescein, spectrophotometer

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

30 minutes

Value:

62.7

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

30 min

Value:

62.7

Results 3

Irritation parameter:

other: mean corrected permeability value

Run / experiment:

30 min

Value:

0.004

Any other information on results incl. tables

For each experiment, the acceptance criteria were fulfilled:

. the individual corneal opacity values of negative controls were < 10,

. the individual OD_{490 nm} values of negative control corneas were < 0.100,

. the solution of fluoresceine (at 5 mg/mL in DPBS) diluted 1:1000 in cMEM had OD_{490 nm} values between 0.850 and 0.940,

. following the 30-minute treatment, the positive control mean in vitro score was 90.3 thus demonstrating the sensitivity of the test system under the experimental conditions of this study.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: scoring table showed above (see section "any other information on materials and methods")

Conclusions:

Under the experimental conditions of this study, according to the mean in vitro scores of the 30 minute treatment, the test item Lead-Copper-Resorcylate-Salycilate (LC 12-15) tested in its original form is classified as irritant to severely irritant for the isolated calf cornea.

Executive summary:

Method:

The corneas were obtained from the eyes of freshly slaughtered calves at the abattoir. They were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. Both compartments of the corneal holder were filled in excesswith Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/streptomycin (cMEM), then the holders were preincubated for 1 hour at 32°C.

Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer (determining the light transmission through the center of each mounted cornea).

 

For the treatment, the test item was used in its original form.

 

The test item was first intended to be tested sequentially in two consecutive experiments, however, according to the results of the first experiment (mean in vitro score at the 30-minute treatment > 55), no second experiment was undertaken and the study was considered as complete.

At the completion of the treatment period, the test item was removed from the front opening of the anterior part of the holder and the epithelium was washed.

The corneas were incubated for 2 hours at. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

After the second opacity measurement, the medium was removed from both compartments of each holder. The posterior compartment was refilled with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS). Then, the holders were incubated vertically for 90 minutes at 32°C.

 

At the end of the 90-minute incubation, the optical density of the solution from the posterior compartment of the holder was measured at 490 nm in order to determine the permeability of the cornea. Then the cornea was removed from the holder and observed for opaque spots and other irregularities.

 

Results

The acceptance criteria were fulfilled and the study was therefore considered to be valid.

No notable opaque spots or irregularities were observed on negative control corneas.

Opacity, residual test item on corneas and fluoresceine fixation were observed on test item-treated corneas.

The mean in vitro score was 62.7.

Conclusion:

Under the experimental conditions of this study, according to the mean in vitro score of the 30‑minute treatment, the test item Lead-Copper-Resorcylate-Salycilate (LC 12-15) tested in its original form is classified as irritant to severely irritant for the isolated calf cornea.