Ferrocholinate

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Reference 2

Reference Type:

publication

Title:

ECVAM international validation study on in vitro tests for acute skin irritation: “Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test”

Author:

Spielmann H, Hoffmann S, Liebsch M, Botham P, Fentem JH, Eskes C, Roguet R, Cotovio J, Cole T, Worth A, Heylings J, Jones P, Robles C, Kandárová H, Gamer A, Remmele M, Curren R, Raabe H, Cockshott A, Gerner I, Zuang V.

Year:

2007

Bibliographic source:

Spielmann H,..., Zuang V. (2007) ECVAM international validation study on in vitro tests for acute skin irritation: “Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test”, Altern Lab Anim., 35 (6): 559-601.

Reference 3

Reference Type:

other: Protocol

Title:

Unnamed

Year:

2018

Report date:

2018

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 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.

Test guideline 2

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 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

In vitro test system

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.

Results and discussion

In vitro

Results

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

Applicant's summary and conclusion

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%).