Calcium fluoride hydroxide phosphate (5 : 0.6-0.8 : 0.2-0.4 : 3)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 12th to April 26th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted October 9th, 2017

Deviations:

no

Principles of method if other than guideline:

Additional information was taken from:
- MatTek Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals, for use with MatTek Corporation’s Reconstructed Human EpiOcularTM Model, 14. July 2014
- Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. “Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test”. Toxicology in Vitro 12, 455-461

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Calcium fluoride hydroxide phosphate (5 : 0.6-0.8 : 0.2-0.4 : 3)

EC Number:

944-884-4

Molecular formula:

Ca5(PO4)3(F)0.6-0.8(OH)0.2-0.4

IUPAC Name:

Calcium fluoride hydroxide phosphate (5 : 0.6-0.8 : 0.2-0.4 : 3)

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: commercially available EpiOcularTM kit. The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: main test: 53.4 mg (tissue 1), 52.5 mg (tissue 2) - additional test: 52.2 mg (tissue 1), 50.7 mg (tissue 2)

POSITIVE AND NEGATIVE CONTROLS
- Amount applied: 50 μl.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

two

Details on study design:

CHEMICALS AND MEDIA
- MTT solution: a MTT stock solution of 5 mg/ml in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 ml of assay medium (resulting in 1 mg/ml). This MTT-solution with the concentration of 1 mg/ml was used in the test.
For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
- DPBS-Buffer - “Dulbecco`s Phosphate Buffered Saline” (DPBS) was used for the rinsing of the tissues. Composition: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 litre and Composition: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 litre. The first buffer was used for rinsing the test item from the tissues. The second buffer was only used for preparing the MTT concentrate.
- MEM with Phenol Red for Pre-Test Serum-free MEM (Minimum Essential Medium)
- Assay Medium for Main Test Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)

PRE-TESTS
- Assessment of Direct Reduction of MTT by the Test Item: the test item was tested for the ability of direct MTT reduction. To test for this ability, 53.4 mg of the solid test item were added to 1 ml of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 ml of MTT solution plus 50 μl of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place and no data correction was necessary.
- Assessment of Coloured or Staining Test Items: 51.6 mg of the test item was added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. As the extract solution was turbid, it was centrifuged (30 seconds at 16 000* g). Then, two 200 μl aliquots of the supernatant and two 200 μl aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 1.792 (>0.08). The test item was possibly interacting with the photometrical measurement and an additional test on colourant controls was performed. The additional test was performed in order to evaluate the amount of colour bound to the tissues. The test item was applied to two additional tissues (= colourant controls) and the test was performed in the same way as described for the main test, but no MTT assay was performed: instead of 300 μl MTT solution, 300 μl assay medium with phenol red was used. The bound colour was extracted and the absorbance of the isopropanol extract was measured in the same fashion as in the MTT assay for coloured test items (without piercing the tissues). As the colourant control result is ≤ 50 % of the viable negative control, a data correction procedure was performed. A non-specific colour in living tissues (NSCliving) control was generated in this case. True tissue viability was calculated as: the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution (%Viabilitytest) minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving), i.e., True tissue viability = [%Viabilitytest] - [%NSCliving].

MAIN TEST
- Preparations: on the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 ml assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 ml fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity for 16 hours.
- Exposure and Post-Treatment: after overnight incubation, the tissues were pre-wetted with 20 μl Ca2+/Mg2+ -free-DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in one- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was tarted. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minuteintervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed at least three times with Ca2+/Mg2+ -free DPBS. Then, the tissues were immediately transferred into 5 ml of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT assay was performed.
- MTT Assay and Extraction: a 24-well-plate was prepared with 300 μl freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 ml isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
- Measurement: the inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μl solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μl isopropanol were also pipetted. The plate was read in a plate spectrophotometer at 570 nm.

EVALUATION CRITERIA
- % Viability > 60 %: no category
- % Viability ≤ 60 %: no prediction can be made (category 1 or 2)

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability %

Value:

98.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD found 1.6, demanded: >0.8 and < 2.5
- Acceptance criteria met for positive control: % mean relative viability of positive control 38.5 %, demanded < 50 % of negative control
- Variation within replicates: 2.9 % (negative control), 4.9 % (positive control), 3.6% (test item), demanded < 20 %
- Range of historical values if different from the ones specified in the test guideline: values for negative control and for positive control were within the range of historical data of the test facility

Any other information on results incl. tables

MEASURED VALUES - MAIN TEST

As blank, the optical density of isopropanol was measured in eight wells of the 96-wellplate. The measured values and their mean are given in the following table:

Table: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.038	0.036	0.035	0.037	0.035	0.037	0.036	0.037	0.036

The absorbance values of negative control, test item and positive control are given in the following table:

Table: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive control	Test material
Tissue 1	1	1.696	0.710	1.695
	2	1.720	0.712	1.699
Tissue 2	1	1.670	0.621	1.644
	2	1.650	0.639	1.630

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol(= corrected values).

Table: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative control	Positive control	Test material
Mean – blank (tissue 1)	1.672	0.675	1.661
Mean – blank (tissue 2)	1.624	0.594	1.601

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

Table: % Viability Positive Control and Test Item

Designation	Positive control	Test material
% Viability (tissue 1)	41.0	100.8
% Viability (tissue 2)	36.0	97.1
% Viability Mean	38.5	99.0

MEASURED VALUES- ADDITIONAL TEST FOR COLOURED TEST ITEMS

As blank, the optical density of isopropanol was measured in eight wells of the 96-wellplate. The measured values and their mean are given in the following table:

Table: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.036	0.037	0.036	0.037	0.036	0.037	0.037	0.038	0.037

The absorbance values of negative control and test item are given in the following table:

Table: Absorbance Values Negative Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Test material
Tissue 1	1	1.774	0.040
	2	1.780	0.039
Tissue 2	1	1.785	0.041
	2	1.766	0.041

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol (= corrected values).

Table: Mean Absorbance Negative Control and Test Item

Designation	Negative control	Test material
Mean – blank (tissue 1)	1.740	0.003
Mean – blank (tissue 2)	1.739	0.004

Comparison of Tissue Viability (Additional Test for Coloured Test Items) For the test item, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative control:

Table: % Tissue Viability

Designation	Test material
% Viability (tissue 1)	0.1
% Viability (tissue 2)	0.2
% Viability Mean	0.2 (rounded value)

% Viability mean main test: 99.0 %

% Viability mean colourant control of additional test: 0.2 %

% Viability corrected: 98.8 %

Applicant's summary and conclusion

Interpretation of results:

other: not classified as eye irritant according to the CLP Regulation (EC) No.1272/2008

Conclusions:

Non eye irritant

Executive summary:

The eye hazard potential of the test material was evaluated according to the OECD 492 in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control.

As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement. Therefore, an additional test for intensely coloured test items was performed. The result of the additional test showed, that the test item colour did not influence the result of the study.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.6. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 38.5 % (< 50 %). Variation within tissue replicates of negative control, positive control and test item was acceptable (< 20 %).

After treatment with the test item, the mean value of relative tissue viability was 98.8 %.

This value is well above the threshold for eye irritation potential (≤ 60 %). Test items that induce values above the threshold are considered non-eye irritant. Under the conditions of the test, Fluorohydroxyapatite is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.