Barium fluoride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-05 - 2017-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to an OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013
* Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 method B.48 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) “Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

June 2015

Test material

Test material information

Constituent 1

Reference substance name:

Barium fluoride

EC Number:

232-108-0

EC Name:

Barium fluoride

Cas Number:

7787-32-8

Molecular formula:

BaF2

IUPAC Name:

barium fluoride

Test material form:

solid: crystalline

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within 2 hours of collection.

SELECTION AND PREPARATION OF EYES FOR THE TEST
* 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 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a 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 so that the appropriate humidity was maintained.

* Eyes examination and acclimatization time
The prepared 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 avoid 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/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. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline 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, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item: 30 mg

Negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three replicates for the test item and the positive control
One replicate for the negative control

Details on study design:

TREATMENT
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea. 30 mg of the test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.
Negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.
Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

TEST ITEM REMOVAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible.Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 4x20 mL saline after treatment

OBSERVATION AND ASSESSMENT OF CORNEAL EFFECTS
The control eyes 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 approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

Experiment I

Value:

1.6

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

Experiment I

Value:

2.7

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

1

Remarks on result:

other: ICE Class II

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

1

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

TEST ITEM
Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse..

POSITIVE CONTROL
The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.
The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

NEGATIVE CONTROL
The negative control (Physiological saline) was classified as non-irritating, UN GHS Classification: Non-classified.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. Therefore this experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with Barium fluoride, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 431 guideline. After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Based on this in vitro eye irritation assay in isolated chicken eyes with Barium fluoride, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.