Caesium nitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-12-07 to 2012-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: testing in compliance with guidelines and CLP

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:

other: OECD guideline for testing of chemicals No. 438, adopted 07 September 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.48, adopted 08.12.2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Caesium nitrate

EC Number:

232-146-8

EC Name:

Caesium nitrate

Cas Number:

7789-18-6

Molecular formula:

Cs.HNO3

IUPAC Name:

caesium nitrate

Test material form:

other: solid

Test animals / tissue source

Species:

other: isolated chicken eyes. species: ROSS 308

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The age and weight of the chickens used for this study were that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 – 2.5 kg). Head collection was performed by a slaughter house technician. The heads were transported to TOXI-COOP ZRT. at the earliest convenience for use approximately within 2 hours from collection. All eyes used in the assay were from the same groups of eyes collected on one specific day.

Test system

Vehicle:

not specified

Controls:

not required

Amount / concentration applied:

The test item was applied in powder form at an amount of 0.03 g to the entire surface of the cornea attempting to cover the cornea surface uniformly. The positive control eyes were treated in a similar way with 0.03 g Imidazole. The negative control eye was treated with 30 μL of saline solution.

Duration of treatment / exposure:

10 s

Observation period (in vivo):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.

Details on study design:

Eyes selection:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2% (v/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes:
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.

Eyes examination and acclimatisation time:
The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ±1.5°C during the acclimatisation and treatment periods.

Identification:
The eyes were identified by chamber number, marked on the door of the chamber.

The base line assessments:
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase by more than 5-7% between the -45 to 60 minutes and the zero time. Slight thinning and changes in thickness (-5% to 3%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effects after treatment. The locations of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.

Treatment:
After the zero reference measurements, the first eye was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied at an amount of 0.03 g by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance while taking care not to damage or touch the cornea with the application equipment.
The positive control eyes were treated in a similar way with 0.03 g Imidazole.
The negative control eye was treated with 30 μL of saline solution.

Test item removal:
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
Residues of Imidazole were still adhering to the corneas surface 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed after the 30 minutes of observation.
The cornea surfaces were not cleared 240 min after the post-treatment rinse.

Observation and assessment of corneal effects:
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Evaluation:
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

corneal swelling 

Run / experiment:

1st experiment (mean 75 min)

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

corneal swelling 

Run / experiment:

2nd experiment (mean 240 min)

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No corrosion/severe irritation effects were observed. A slight irritation potential of the test item was observed.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with cesium nitrate, no ocular corrosion or severe irritation potential was observed. Thus, according to the guideline OECD 438, the test item cannot be classified as an ocular corrosive or severe eye irritant. Furthermore, the results suggest that the test item was not irritating.

Executive summary:

The in vitro Isolated Chicken Eye Test (ICET) was perfomed according to OECD guideline 438 and EU method B.48 to evaluate the potential ocular corrosivity or severe irritancy of the test item cesium nitrate. The test compound was applied in its powdered form via a single dose of 0.03 g onto the cornea of isolated chicken eyes.

During the testing no ocular corrosion or severe irritation potential of the test item was observable. Thus, according to the guideline OECD 438, cesium nitrate cannot be classified as an ocular corrosive or severe eye irritant. Furthermore, the results suggest that the test item was not irritating. However, to obtain a definitive classification in relation to the irritation potential, a further in vivo rabbit study is required.