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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 November to 10 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium tartrate
EC Number:
221-621-5
EC Name:
Calcium tartrate
Cas Number:
3164-34-9
Molecular formula:
C4H6O6.Ca
IUPAC Name:
Calcium carbonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): TARTRATE DE CALCIUM
- Physical state: amber powder
- Analytical purity: 93.44%
- Lot/batch No.: CAMP 09-10
- Expiration date of the lot/batch: December 2011
- Storage conditions of test material: at room temperature, protected from light and humidity.

Test animals / tissue source

Species:
other: bovine calf
Details on test animals or tissues and environmental conditions:
Calf eyes were obtained from freshly slaughtered calves at the abattoir SOCAVIA, Cany Barville, France.
Calf corneas are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Storage of the cornea: the corneas 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: N/A
Amount / concentration applied:
100%
Duration of treatment / exposure:
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.

Assembly of the corneas and the holders
The corneas (conserved 24 hours) were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the
holder.
The anterior half of the holder was then positioned on top of the cornea and fixed in place with screws.
Each cornea was identified with the corresponding holder number.

Preincubation
Both compartments of the corneal holder were filled in excess with Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/
streptomycin (cMEM) at room temperature.
The holders were plugged and preincubated horizontally for 1 hour at 32°C in a water bath.

Treatment of corneas
Three corneas were used for each treated series (test item, positive control and negative control).
In order to respect the treatment times rigorously, each operation (treatment, rinsing, measurement…) was carried out per three corneas from the
same series and always in the same order.

At the end of the 1-hour preincubation period the opacity of the cornea was measured.
After the first opacity measurement, the medium was removed from both compartments of the holder using a metal gavage tube attached to a
vacuum pump to ensure complete evacuation, then the posterior compartment was refilled with fresh cMEM previously heated at 32°C.
Observation period (in vivo):
Incubation after treatment
Following the 30-minute treatment, the corneas were incubated vertically (holders placed horizontally) for 2 hours in a water bath at 32°C after the
rinsing of the dosage form. At the completion of the 2-hour incubation period, the second opacity measurement was performed.
Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the
dosage form.

Opacity measurement
The measurement of the opacity was performed using an opacitometer. The opacitometer determined the light transmission through the center of
each mounted cornea. A numerical opacity (arbitrary unit) was displayed and recorded. The opacity of each cornea was measured at two occasions in the study:
- just before the treatment, the value is called OPT0,
- 2 hours after the end of the treatment period (or directly after the end of the treatment period for the 4-hour treatment), the value is called OPT2.

Just before each time of measurement (OPT0 and OPT2), the opacitometer was calibrated using specific calibrators. Values obtained for each
calibrator must be as follows:
- calibrator No. 1: 75,
- calibrator No. 2: from 145 to 155,
- calibrator No. 3: from 218 to 232.

Just after each time of measurement (OPT0 and OPT2), the calibrator No. 1 was measured. The value obtained fell between 73 and 77.

The opacity of the corneas was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus a holder
containing only cMEM and placed in the left hand chamber.
Number of animals or in vitro replicates:
N/A
Details on study design:
Permeability determination
After the second opacity measurement (OPT2), the medium was removed from both compartments of each holder using metal gavage tube attached
to a vacuum pump to ensure complete evacuation. The posterior compartment was refilled with cMEM at 32°C, while the anterior compartment
received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate Buffered Saline (DPBS).

For each series of three corneas, a chronometer started from the fluoresceine application time of the first cornea of the series.
Then, the holders were incubated vertically in a water bath at 32°C for 90 minutes.

At the end of the 90-minute incubation, the maximum of solution recoverable from the posterior compartment of the holder was transferred in
identified tubes using a single use plastic syringe and a needle.
After homogenization of the solution, its Optical Density (OD) was measured at 490 nm using identified single use cuvette of 1 cm path length and a
spectrophotometer blanked with cMEM. Any solutions giving an OD490 nm beyond the linear range (DO > 1.000) of the spectrophotometer were
diluted 1:4 in cMEM.
The solution of fluoresceine used (at 5 mg/mL in DPBS) was diluted 1:1000 in cMEM and the OD490 nm of this dilution was measured. As the value
obtained was between 0.850 and 0.940, the experiment was validated.

Sampling of tissues
After the sampling of the medium in the posterior compartment for permeability determination, corneas were removed from the holders and
observed for opaque spots or other irregularities.
Any separation of the epithelium, as any other observations, was recorded.
The corneas were then fixed in 10% neutral buffered formalin for at least 24 hours at room temperature.

Histological examination
No microscopic examinations were performed and the corneas were destroyed at the finalization of the study report.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: BCOP opacity
Run / experiment:
30 min
Value:
>= 0 - <= 0.3
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation parameter:
other: BCOP permeability
Run / experiment:
30 min
Value:
>= 0.024 - <= 0.041
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation parameter:
other: BCOP opacity
Run / experiment:
4 h
Value:
>= 1.7 - <= 2.7
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation parameter:
other: BCOP permeability
Run / experiment:
4 h
Value:
>= 0.003 - <= 0.01
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified

In vivo

Irritant / corrosive response data:
Observation of corneas after treatment
No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4-hour treatment.

No notable opaque spots or irregularities were observed on test item-treated corneas following the 30-minute treatment while residual test item was observed on all corneas after the 4 hour treatment.

Evaluation of the results
Following the 30-minute treatment, the mean in vitro score was 0.4. Then following the 4-hour treatment, the mean in vitro score was 1.7.

Applicant's summary and conclusion

Interpretation of results:
slightly irritating
Remarks:
Migrated information minimal possible grade in this test Criteria used for interpretation of results: expert judgment
Conclusions:
According to both mean in vitro scores of the 30 minute and 4-hour treatments, the test item, TARTRATE DE CALCIUM, tested in its original form is classified as slightly irritant for the isolated calf cornea.
This does not correspond to a classification requirement of CLP Regulation.
Executive summary:

The aim of this study was to evaluate the potential irritant properties of the test item, TARTRATE DE CALCIUM, for the isolated calf cornea.

This study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practices.

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 excess with 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 tested in one experiment using treatment durations of 30 minutes and 4 hours.

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.

Following the 30-minute treatment, the corneas were incubated for 2 hours at. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the dosage form.

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.

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

For each treatment time, 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, either following the 30-minute treatment or following the

4-hour treatment.

No notable opaque spots or irregularities were observed on test item-treated corneas following the 30-minute treatment while residual test item was observed on all corneas after the 4‑hour treatment.

Following the 30-minute treatment, the meanin vitro score was 0.4. Then following the 4-hour treatment, the meanin vitroscore was 1.7.

Conclusion

According to both meanin vitro scores of the 30‑minute and 4-hour treatments, the test item, TARTRATE DE CALCIUM, tested in its original form is classified as slightly irritant for the isolated calf cornea.