Ferrocholinate

Eye irritation

Administrative data

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)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

other: Standard Operating Procedure (SOP)

Title:

Unnamed

Year:

1999

Report date:

1999

Reference 3

Reference Type:

other: Protocol

Title:

Unnamed

Year:

1996

Report date:

1996

Reference 4

Reference Type:

other: Standard Operating Procedure (SOP)

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

Ferrocholinate

EC Number:

215-649-7

EC Name:

Ferrocholinate

Cas Number:

1336-80-7

Molecular formula:

C11H24FeNO11

IUPAC Name:

2-hydroxyethyl(trimethyl)azanium;iron(3+);2-oxidopropane-1,2,3-tricarboxylate;trihydrate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Yellow-green powder
- Purity: 12.2 % Ferric
- Homogeneity: Homogeneous
- Molecular Weight: 941.56 g/mol

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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

Applicant's summary and conclusion

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.