Ferrocholinate

Endpoint summary

Description of key information

SKIN IRRITATION:

Relative tissue viability > 50%: non-irritant to skin.

Relative tissue viability <= 50%: irritant to skin.

The mean value of relative tissue viability of the test item was reduced to 68.1%. The test item is considered non-irritant to skin (OECD Guideline 439).

EYE IRRITATION:

IVIS > 3 and ≤ 55: induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only.

The calculated mean IVIS of the test item was 32.67. The test item is neither UN GHS Classification Category I nor No Category (OECD Guideline 437).

The test item is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test (OECD Guideline 492).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24. Oct. 2019 (Study Plan dated); 05. Nov. 2019 (Experimental Starting Date); 08. Nov. 2019 (Experimental Completion Date)

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:

Adopted 18. Jun. 2019

Deviations:

yes

Remarks:

The pre-incubation time should be 18-24 hours. Due to delayed delivery of the test system the pre-incubation time was shortened. This can be seen as uncritical because according to the supplier the pre-incubation must be at least one hour.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

human skin model EpiDermTM

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

not specified

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 05. Nov. 2019
Batch no.: 30834

PERFORMANCE OF THE STUDY

1. PRE-TESTS
- Assessment of Coloured or Staining Test Items
It was tested whether the test item develops a colour without MTT addition. 26.9 mg test item were giv-en in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.
- 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, 27.2 mg test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. Untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.

2. PRE-INCUBATION OF TISSUES
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 1% CO2 and ≥ 95% relative humidity for 1 hour.
After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay me-dium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 2 hours and 20 minutes.

3. TREATMENT
One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a ny-lon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.
The following amounts of test item were applied to the tissues: 26.5 mg (tissue 1), 26.8 mg (tissue 2) and 27.3 mg (tissue 3).
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then trans-ferred into a new 6-well-plate with fresh assay medium (0.9 mL). The surface of the inserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 23 hours and 20 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

4. MEDIUM RENEWAL
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 25 minutes for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

5. MTT ASSAY
After a total incubation time of 42 hours and 45 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution (1 mg/ml) in each well. The tissues were blotted on the bottom and then trans-ferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and 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-well-plate which was read in a plate spectrophotometer at 570 nm.


EVALUATION

The values of the 96-well-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded val-ues may lead to slightly different results

1. CALCULATION
Calculations were performed as follows:
Calculation of mean OD of the blank isopropanol (ODBlk)
Subtraction of mean ODBlk of each value of the same experiment (corrected values)
Calculation of mean OD of the two replicates for each tissue
Calculation of mean OD of the three relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100% viability

The photometric absorbance of the negative controls is considered as 100%. For each replicate of test item and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:
% tissiue viability = [ODreplicate test item resp. positive control / ODmean of negative controls]*100

2. ASSESSMENT
If the % of tissue viability is <= 50% of negative control: Corrosive/Irritant to skin (UN GHS Category 1 or 2)
If the % of tissue viability is > 50% of negative control: Non-irritant to skin (No Category for Skin Irritation)

FINDINGS ANS RESULTS

1. ASSESSMENT AND VALIDITY
The mean value of relative tissue viability of the test item was reduced to 68.1 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.

All validity criteria were met:
- OD of negative control
Demanded: >= 0.8 and <= 2.8
Found: 1.8
- % tissue viability of positive control SDS
Demanded: <= 20% of negative control
Found: 3.3%
- SD of mean viability of the tissue replicates (%)
Demanded: <= 18%
Found: 7.8% (negative control), 0.3% (positive control) and 6.1% (test item)

Values for negative control and for positive control were within the range of historical data of the test facility:
- Negative control (OD):
Substance: DPBS buffer
Mean: 1.780
Standard deviation: 0.312
Range: 0.476 – 2.471
Study: 1.766
- Positive control (% OD compared to Negative Control)
Substance: Sodium Dodecyl Sulphate Solution 5%
Mean: 4.2%
Standard deviation: 2.9%
Range: 1.7 – 17.1%
Study: 3.3%

Therefore, the experiment is considered valid.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts of test material applied to each tissue:
Tissue 1: 26.5 mg
Tissue 2: 26.8 mg
Tissue 3: 27.3 mg

NEGATIVE CONTROL
- Amounts applied: Each tissue was treated with 30 µL DPBS buffer.

POSITIVE CONTROL
- Amounts applied: Each tissue was treated with 30 µL 5% SDS-solution

Number of replicates:

Three replicates.

Irritation / corrosion parameter:

% tissue viability

Value:

68.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Ferric Choline Citrate is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method (OECD Guideline 439), since after the treatment, the mean value of relative tissue viability was reduced to 68.1% (this value is above the threshold for skin irritation <=50%).

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:

19. Nov. 2019 (Study Plan dated); 21. Nov. 2019 (Experimental Starting Date); 21. Nov. 2019 (Experimental completation Date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 437 (EU Method B.47)

Qualifier:

according 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)

Version / remarks:

Version 09. Oct. 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Version 14. Feb. 2017. EU Method B.47 (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).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160

Version / remarks:

Version 25. Oct. 2011. “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany.
- Characteristics of donor animals: The cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 20 minutes.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: 20%

VEHICLE
- Concentration: 10-fold concentrated HBSS solution, which was previously diluted in demin. water (1:10).
- Batch no.: T20191121

Duration of treatment / exposure:

Incubation time: 4h

Number of animals or in vitro replicates:

For each treatment group (negative control solution, test item solution or positive control), three replicates were used.

Details on study design:

CONDUCT OF THE STUDY

PREPARATIONS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

EXPERIMENTAL PARAMETERS
Date of treatment: 21. Nov. 2019
Incubation time: 4 h
Negative control: HBSS
Positive control: imidazole, 20 % solution in HBSS

METHOD DESCRIPTION
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item solution or positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, 750 µL test item solution or positive control solution were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the test item, the following treatment procedure was performed:

- Closed Chamber Method
The respective substance (negative control solution, test item solution or positive control soltuion) was applied by pipetting 750 µL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium that the cornea was evenly covered.
Exposure time of the controls and the test item on the corneas was 4 hours at 32 ± 1 °C.
After thorough rinsing the anterior chambers with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phenol red and the final opacity value of each cornea was recorded.
After post-incubation time, the cMEM without phenol red was renewed in both chambers of each cornea holder. Then, the final opacity value of each cornea was recorded.

- Permeability Test
After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluorescein solution was added to the front chamber of each cornea holder for the detection of permeability of the corneas.
For solid non-surfactant test items, a sodium fluorescein solution with a concentration of 5 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.
 
EVALUATION

CALCULATION OF OPACITY
The change of opacity value of each treated cornea with test item, positive control or negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
Opacity = [(Io/I)-b]/a
a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
Io = the empirically determined illuminance through a cornea holder with windows and
Medium, here: Io= 1097.17
I = the measured illuminance (unit: LUX)

CALCULATION OF PERMEABILITY
The corrected OD492 value of each cornea treated with test item or positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control or negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

CALCULATION OF IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.


VALIDITY
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Irritation parameter:

in vitro irritation score

Value:

32.67

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

other: Neither Category I nor No Category

Conclusions:

The test item Ferric Choline Citrate showed effects on the cornea of the bovine eye. The calculated mean IVIS was 32.67.
According to OECD Guideline no. 437 (Oct. 2017), the test item Ferric Choline Citrateis is neither UN GHS Classification Category I (did not induce serious eye damage) nor No Category. According to this guideline, a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23. Oct. 2019 (Study Plan dated); 05. Nov. 2019 (Experimental Starting Date); 08. Nov. 2019 (Experimental Completion Date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

18. Jun. 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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.

Source: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 05. Nov. 2019
Batch no.: 30633

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

55.2 mg in replicate 1 and 53.4 mg in replicate 2.

Duration of treatment / exposure:

The test item was appliad to the human cornea tissue model for an exposure time of 6 hours.

Duration of post- treatment incubation (in vitro):

For post-treatment incubation, the tissues were incubated for 18 hours.

Number of animals or in vitro replicates:

Two replicates.

Details on study design:

PERFORMANCE OF THE STUDY

PRE-TESTS
- Assesment 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, 53.2 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 ≥ 95% 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
53.5 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. Then, two 200 µL aliquots of the resulting solution 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 0.0170 (≤ 0.08). Therefore, the main test was performed without colourant controls.

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 ≥ 95% 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 ≥ 95% relative humidity for 21 hours and 17 minutes.
- Exposure and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and a defined amount of the test item (52.2 mg in replicate 1 and 534 mg in replicate 2) were applied in duplicate in one- minute- intervals.
At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with 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 ≥ 95% 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 ≥ 95% 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-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
 
RhCE TISSUE CONSTRUCT USED
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.
Source: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 05. Nov. 2019
Batch no.: 30633

DOSES OF TEST CHEMICAL AND CONTROL SUBSTANCES USED
Test chemical: 55.2 mg in replicate 1 and 53.4 mg in replicate 2.
Negative control: Sterile demineralised water
Positive control: Methyl acetate (C3H6O2, CAS No. 79-20-9)

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded val-ues may lead to slightly different results.
- Calculation
Calculation of mean OD of the blank isopropanol (ODBlk)
Subtraction of mean ODBlk of each value of the same experiment (corrected values)
Calculation of mean OD of the two replicates for each tissue
Calculation of mean OD of the two relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability
To calculate the relative tissue viability, the following equation was used:
% Viability = (OD corrected of test item or positive control / OD corrected of mean negative control)*100

VALIDITY
Values for negative control and for positive control were within the range of historical data of the test facility.
Historical data - Optical density (negative control: demineralised water): Exposure time 6h, Mean 1.647, Standard deviation 0.249, Range 1.047-2.340
Study - Optical density (negative control: demineralised water): 1.717
Historical data - Relative Tissue Viability (positive control: methyl acetate): Exposure time 6h, Mean 34.9%, Standard deviation 7.0%, Range 21.1-53.9%
Study - Relative Tissue Viability (positive control: methyl acetate): 29.5 %
Therefore, the experiment is considered valid.

Irritation parameter:

other: Relative tissue viabilaty

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

other: Either eye irritant or inducing serious eye damage

Conclusions:

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Under the conditions of the test, Ferric Choline Citrate is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

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:

The test item Ferric Choline Citrate is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method, since after the treatment, the mean value of relative tissue viability was reduced to 68.1% (this value is above the threshold for skin irritation <=50%).  

EYE IRRITATION:

The test item Ferric Choline Citrate showed effects on the cornea of the bovine eye. The calculated mean IVIS was 32.67. According to OECD Guideline 437 (Oct. 2017), the test item Ferric Choline Citrateis is neither UN GHS Classification Category I (did not induce serious eye damage) nor No Category. According to this guideline, a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required. Under the conditions of the test, Ferric Choline Citrate is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

Considering both tests (OECD Guideline 437 and OECD Guideline 492), Ferric Choline Citrate is classified as irritant to eye.