Barium fluoride

Endpoint summary

Description of key information

Barium fluoride appeared to be not irritating to the skin, while it induced eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-12 - 2017-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Version 29 July 2016
* Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: three-dimensional human epidermis model

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international validation study and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: three-dimensional human epidermis model
- Tissue batch number(s): 17-EKIN-025
- Expiration date: 26 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature (24.8-26.9°C)

APPLICATION
The plates with the treated epidermis units were incubated for the exposure time of 4 hours (± 10 min).

REMOVAL OF TEST MATERIAL
After the 4 hour incubation time, the EPISKINTM(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

MTT TEST
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light, in a >95% humidified atmosphere.

FORMAZAN EXTRACTION
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
For more than 2 disks:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5.
The acceptable mean percentage viability range for positive controls is < 20%.
Control OD values should be within the historical control range.
The difference of viability between the two tissue replicates should not exceed 30%
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM
As the test item was solid, 20 mg of test item was applied evenly to the epidermal surface of each of two test item treated units and then 100 µL physiological saline was added to the test item to ensure good contact with the epidermis.

NEGATIVE AND POSITIVE CONTROLS
50 μL of negative control (Physiological saline) or positive control (Glacial acetic acid) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

Duration of treatment / exposure:

4 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Value:

100.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.004, Non-specific Colour % was calculated as 0.5%. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1 below (Any other information on results incl. tables). The OD values for the test item treated skin samples showed 100.4% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The two positive control treated tissues showed 0.3% viability demonstrating the proper performance of the assay.
One individual opacity density data (0.000) and the mean value (0.003) of the positive control in the corrosivity testing were slightly lower than the lower limit of the historical control range (0.005). These facts had no impact on the results or integrity of the study since the positive control material showed severe effect.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 2.9%.
The difference of viability between the two negative control tissue samples in the MTT assay was 0.3%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1. Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control :	1	0.808	0.761	100.2
Physiological saline (0.9% (w/v) NaCl)	2	0.806	0.758	99.8
	mean	-	0.760	100.0
Positive Control :	1	0.053	0.005	0.7
Glacial acetic acid	2	0.047	0.000	0.0
	mean	-	0.003	0.3
Test Item :	1	0.799	0.752	99.0
Barium fluoride	2	0.821	0.774	101.9
	mean	-	0.763	100.4

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

GHS criteria not met

Conclusions:

Barium fluoride is not corrosive to the skin.

Executive summary:

An in vitro skin irritation test of Barium fluoride was performed in a reconstructed human epidermis model EPISKIN (SM) according to the OECD No. 431 guideline.

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline(0.9% (w/v) NaCl solution) treated epidermis were used as negative controls and glacial acetic acid treated epidermis were used as positive controls (two units/control). Two additional disks were used to provide an estimate of colour contribution (NSC_living) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability is < 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Barium fluoride, the mean cell viability was 100.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-12 - 2017-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Version 28 July 2015
* Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation
Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
* EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of
the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: three-dimensional human epidermis model

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: three-dimensional human epidermis model
- Tissue batch number(s): 17-EKIN-025
- Expiration date: 26 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature (26.4-27.6°C)
- Temperature of post-treatment incubation: 37°C

APPLICATION
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min).

REMOVAL OF TEST MATERIAL
After the 15 minutes incubation time, the EPISKINTM(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2 in a 95% humified atmosphere.

MTT TEST
After the 42 hours incubation, MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light, in a >95% humidified atmosphere.

FORMAZAN EXTRACTION
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
Mean tissue viability % is ≤ 50%: Category 2 or Category 1
Mean tissue viability % is > 50%: Non-Irritant

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
The SD calculated from individual % tissue viability values of the three test item treated replicates should be ≤ 18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 20 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

NEGATIVE AND POSITIVE CONTROLS
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

79.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As no colour change was observed after three hours of incubation of the test item in MTT working solution, thus the test item did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.
As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.007, Non-specific Colour % was calculated as 1.0%. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1 below (Any other information on results incl. tables). The OD values for the test item treated skin samples showed 79.5% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the three negative control tissues was in the recommended range (0.720). Standard deviation of the viability results for negative control samples was 2.5%.
The positive control treated tissues showed 6.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 2.4%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1. Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control :	1	0.752	0.706	98.0
Phosphate buffered saline	2	0.761	0.715	99.2
	3	0.787	0.740	102.8
	mean	-	0.720	100.0
Positive Control :	1	0.086	0.040	5.5
5% (w/v) SDS* solution	2	0.094	0.047	6.5
	3	0.101	0.054	7.5
	mean	-	0.047	6.5
Test Item :	1	0.638	0.591	82.1
Barium fluoride	2	0.616	0.569	79.0
	3	0.604	0.558	77.4
	mean	-	0.573	79.5

* SDS: Sodium Dodecyl Sulphate

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

GHS criteria not met

Conclusions:

Barium fluoride is not irritating to the skin.

Executive summary:

An in vitro skin irritation test of Barium fluoride was performed in a reconstructed human epidermis model EPISKIN (SM) according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with Barium fluoride, the mean cell viability was 79.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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

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

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

1

Remarks on result:

other: ICE Class II

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.

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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-18 to 2010-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

, 2002-04-24

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan, Belton, Leics, England
- Age at study initiation: 9 - 11 weeks old
- Weight at study initiation: 2.210 kg - 2.261 kg
- Housing: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least three times a week.
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.8 – 23.4ºC
- Relative humidity: 38 - 81%
- Air changes: Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): On average 99.5 mg (range 98.9 - 99.9 mg) of the test substance was installed in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

No data

Observation period (in vivo):

Approximately 1, 24, 48 and 72 hours and 7 and 14 days (all animals) and 21 days (two animals) after instillation of the test substance.

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later, after considering the degree of eye irritation observed in the first animal.

SCORING SYSTEM: draize scoring system was used. In cases of equivocal results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety Commission, Washington, D.C. 20207 was used for additional control purposes.

TOOL USED TO ASSESS SCORE: Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp.

OBSERVATIONS.
Mortality/Viability: Twice daily
- Toxicity: At least once daily
- Body weight: Day of treatment (prior to instillation) and after the final observation
Necrospy: no necropsy was performed.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

The corneal injury consisted of opacity (maximum grade 1: Scattered or diffuse areas of opacity, details of iris clearly visible) and epithelial damage (maximum 10, 15 or 20% of the corneal area). Slight dulling of the normal luster of the cornea was also observed. The corneal injury fully resolved within 72 hours in two animals and within 7 days in the other animal.
Iridial irritation grade 1(Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive)) was observed and resolved within 24 hours in one animal and within 48 hours in the other two animals.
The irritation of the conjunctivae consisted of redness (maximum grade 3: Diffuse beefy red), chemosis (maximum grade 3: Swelling with lids about half closed) and discharge (Discharge with moistening of the lids and hairs just adjacent to lids to any amount different from normal and/or lacrimation) and completely resolved within 14 days in one animal and within 21 days in the other two animals.
There was no evidence of ocular corrosion.
No staining of (peri) ocular tissue by the test substance was observed and no test substance remnants were seen.

Other effects:

Toxicity/Mortality:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

According to the Regulation (EC) No 1272/2008, barium chloride dihydrate should be classified as Irritating to eyes (Category 2).
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is classified as eye irritant ("irritating to eyes")

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Based on the results of an in vitro eye irritation assay in isolated chicken eyes, barium fluoride is not classified as a severe irritant and not classified as non-irritant. Since information is required to conclude on the classification, an in-vivo eye irritation study with the substance barium chloride was used in an analogue approach. Barium chloride was identified as an analogue of barium fluoride based on the presence of barium as the common cation.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal#2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal#2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal#2

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal#2

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal#3

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal#3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal#3

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal#3

Time point:

24/48/72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible within: 14 days

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on read-across from barium chloride, the substance barium fluoride is considered as irritating to eyes. Since the effects are not reversible within 7 days, subcategory 2A is applied according to the GHS UN criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro EPISKIN^TM(SM) model test, barium fluoride is non-corrosive and non-irritant to skin. Therefore no classification for skin irritation is warranted according to Regulation (EC) 1272/2008 (CLP).

Eye irritation:

Based on the results of an in vitro eye irritation assay in isolated chicken eyes, barium fluoride is not classified as a severe irritant and not classified as non-irritant. Since information is required to conclude on the classification, an in-vivo eye irritation study with the substance barium chloride was used in an analogue approach. Based on the results of this study, it is concluded that barium chloride is irritating to eyes (Cat. 2; H319 "Causes serious eye irritation"). The same classification is applied for barium fluoride.